CN1250721C - Dimeric thrombopoietin peptide mimetics binding to MP1 receptor and having thrombopoietic activity - Google Patents

Dimeric thrombopoietin peptide mimetics binding to MP1 receptor and having thrombopoietic activity Download PDF

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CN1250721C
CN1250721C CN 99812517 CN99812517A CN1250721C CN 1250721 C CN1250721 C CN 1250721C CN 99812517 CN99812517 CN 99812517 CN 99812517 A CN99812517 A CN 99812517A CN 1250721 C CN1250721 C CN 1250721C
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C·-F·刘
U·菲格
J·奇塔姆
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安姆根有限公司
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/524Thrombopoietin, i.e. C-MPL ligand
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04Linear peptides containing only normal peptide links
    • C07K7/06Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04Linear peptides containing only normal peptide links
    • C07K7/08Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/70Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Abstract

本发明涉及化合物领域,特别是具有血小板生成活性的肽或多肽。 The present invention relates to the field of compounds, especially peptides or polypeptides having a platelet producing activity. 本发明的肽或多肽可用于哺乳动物,升高血小板或血小板前体(如巨核细胞)的数量。 Peptide or polypeptide of the present invention may be used in a mammal, raise the number of platelets or platelet precursors (e.g., megakaryocytes) in.

Description

与MPl受体结合并具有血小板生成活性的模拟二聚体血小板生成素肽 MPl receptor binding activity and having thrombopoietic analog peptide dimer thrombopoietin

本申请要求1998年10月23日提交的、美国临时申请06/105 348的优先权。 This application claims the 1998 October 23 filed US Provisional Application No. 06/105 348.

发明领域基本上,本发明涉及化合物领域,特别是具有血小板生成活性的肽或多肽。 FIELD OF THE INVENTION Basically, the present invention relates to the field of compounds, especially those with active thrombopoietic peptide or polypeptide. 本发明的肽或多肽可用于哺乳动物,增加血小板或血小板前体(如巨核细胞)的产量。 Peptides or polypeptides of the invention can be used in mammals, increase production platelets or platelet precursors (e.g., megakaryocytes) in.

发明背景本发明涉及化合物领域,特别是具有体外和体内刺激血小板及其前体细胞如巨核细胞产生能力的肽。 Background of the Invention The present invention relates to the field of compounds, especially in vivo and in vitro, and stimulation of platelet precursor cells such as megakaryocytes peptide-producing ability. 在探讨本发明化合物的性质之前,下面将提供两种具有血小板生成活性的蛋白质:血小板生成素(TPO)和巨核细胞生长发育因子(MGDF)作为背景资料。 Before discussing the nature of the compounds of the present invention, the following will offer two proteins having thrombopoietic activity: thrombopoietin (TPO) and megakaryocyte growth and development factor (of MGDF) as background information.

内源性血小板生成素(TPO)的克隆(Lok等,自然,369:568-571(1994);Bartley等,细胞,77:1117-1124(1994);Kuter等,美国科学院院刊,91:11104-11104(1994);de Sauvage等,自然,369:533-538(1994);Kato等,生物化学杂志,119:229-236(1995);Chang等,生物化学杂志,270:511-514(1995))迅速加深了我们对巨核细胞生成(巨核细胞的产生)和血小板生成(血小板的产生)的理解。 Clone (Lok et endogenous thrombopoietin (TPO), nature, 369: 568-571 (1994); Bartley et al., Cell, 77: 1117-1124 (1994); Kuter et al., Proc Natl Acad Sci USA, 91: 11104-11104 (1994); de Sauvage et al., Nature, 369: 533-538 (1994); Kato et al., Journal of Biological Chemistry, 119: 229-236 (1995); Chang et al., Journal of Biological Chemistry, 270: 511-514 (1995)) rapidly deepen our understanding of megakaryocytes generate (produce megakaryocytes) and platelet production (platelet production).

人内源性TPO是一种60-70kDa的糖基化蛋白,主要在肝脏和肾脏产生,包括332个氨基酸(Bartley等,细胞,77:1117-1124(1994);Chang等,生物化学杂志,270:511-514(1995))。 Human Endogenous TPO is glycosylated 60-70kDa of a protein, mainly in the liver and kidney, comprising 332 amino acids (Bartley et al., Cell, 77: 1117-1124 (1994); Chang et al., Journal of Biological Chemistry, 270: 511-514 (1995)). 该蛋白在不同物种间高度保守,与人促红细胞生成素在氨基末端(第1到172氨基酸)(Bartley等,细胞,77:1117-1124(1994))有23%的同源性(Gurney等,血液,85:981-988(1995))。 The proteins are highly conserved across species, the human erythropoietin amino terminus (amino acids of 1-172) (Bartley et al., Cell, 77: 1117-1124 (1994)) have 23% homology (Gurney et , blood, 85: 981-988 (1995)). 业已显示,内源性TPO拥有血小板生成的关键生物调节子的所有特性。 It has been shown that endogenous TPO has all the characteristics of the key biological regulator of platelet production promoter. 其体外作用包括,特异性诱导纯化鼠骨髓造血干细胞(Zeigler等,血液,84:4045-4052(1994))和人CD34+细胞(Lok等,自然,369:568-571(1994);Rasko等,干细胞,15:33-42(1997)),巨核细胞集落形成,巨核细胞的产生呈倍性增加(Broudy等,血液,85:402-413(1995)),并诱导巨核细胞的终末成熟和血小板产生(Zeigler等,血液,84:4045-4052(1994);Choi等,血液,85:402-413(1995))。 Vitro effects which include specific induction of purified murine hematopoietic stem cells (Zeigler et al., Blood, 84: 4045-4052 (1994)) and human CD34 + cells (Lok et al., Nature, 369: 568-571 (1994); Rasko et al., Stem cells, 15: 33-42 (1997)), megakaryocyte colony formation, produce megakaryocytes were fold increase (Broudy et al., blood, 85: 402-413 (1995)), and to induce megakaryocyte terminal maturation and platelet production (Zeigler et al., blood, 84: 4045-4052 (1994); Choi et al., blood, 85: 402-413 (1995)). 相反,针对TPO受体(c-Mpl)的合成反义寡脱氧核苷酸可以显著抑制巨核细胞祖细胞的集落形成能力(Methia等,血液,82:1395-1401(1993))。 In contrast, for the TPO receptor (c-Mpl) Synthesis of Antisense oligodeoxynucleotides current can be remarkably suppressed megakaryocyte progenitor cell colony forming ability (Methia et al., Blood, 82: 1395-1401 (1993)). 此外,敲除c-Mpl基因的小鼠患有严重的血小板缺乏症,并缺乏巨核细胞(Alexander等,血液,87:2162-2170(1996))。 Moreover, c-Mpl knockout mice gene with severe thrombocytopenia, and lack of megakaryocytes (Alexander et al., Blood, 87: 2162-2170 (1996)).

重组人MGDF(rHuMGDF,Amgen Inc.,Thousand Oaks,加利福尼亚)是另一种与TPO相关的血小板生成多肽。 Recombinant human MGDF (rHuMGDF, Amgen Inc., Thousand Oaks, CA) is another thrombopoietic related to TPO polypeptide. 该物质是通过应用质粒转化的大肠杆菌生产的,所述质粒含有编码包括人TPO氨基末端受体结合域的截断蛋白的cDNA(Ulich等,血液,86:971-976(1995))。 This material is obtained by application of the plasmid transformed E. coli produced a plasmid comprising cDNA which encodes human TPO amino-terminal receptor binding domain of the truncated protein (Ulich et al., Blood, 86: 971-976 (1995)). 该多肽被提取、再包装、纯化,并在其氨基末端与聚(乙烯二醇)(PEG)部分共价连接。 The polypeptide is extracted, re-packaging, purified and covalently attached to the amino terminus of poly (ethylene glycol) (PEG). 获得的分子这里称为PEG-rHuMGDF或简称为MGDF。 The resulting molecule is referred herein simply referred to as PEG-rHuMGDF or MGDF.

应用动物模型的很多试验(Ulich,TR等,血液,86:971-976(1995);Hokom,MM等,血液,86:4486-4492(1995))业已明确显示TPO和MGDF在骨髓移植和血小板缺乏症治疗中的疗效,而后一种情况通常由于化疗或放疗所致。 Many experimental animal model of application (Ulich, TR et al., Blood, 86: 971-976 (1995); Hokom, MM et al., Blood, 86: 4486-4492 (1995)) has been clearly demonstrated TPO and MGDF in bone marrow transplantation and platelets lack of efficacy in the treatment of disease, the latter usually due to chemotherapy or radiation-induced. 在人类获得的初步资料已证实应用MGDF可以在多数情况下提高血小板计数(Basser等,柳叶刀,348:1279-81(1996);Kato等,生物化学杂志,119:229-236(1995);Ulich等,血液,86:971-976(1995))。 Preliminary data obtained in humans has been shown to improve application MGDF can in most cases platelet count (Basser et al., Lancet, 348: 1279-81 (1996); Kato et al., Journal of Biological Chemistry, 119: 229-236 (1995) ; Ulich et al., blood, 86: 971-976 (1995)). MGDF还可用于增强血小板捐供,因为给健康血小板供者应用MGDF,可以将循环血小板数量提高到大约原始数值的3倍。 MGDF may also be used for enhancing platelet donation, as application to healthy platelet donors MGDF, the platelet number of cycles can be increased to about 3 times the original value.

TPO和MGDF通过与c-Mpl受体结合发挥作用,该受体主要在某些造血细胞表面表达,如巨核细胞,血小板,CD34+细胞和原始祖细胞(Debili,N.等,血液,85:391-401(1995);de Sauvage,FJ等,自然,369:533-538(1994);Bartley,TD等,细胞,77:1117-1124(1994);Lok,S.等,自然,369:565-8(1994))。 TPO and MGDF acts by binding to c-Mpl receptor, which receptors are expressed primarily in the surface of certain hematopoietic cells, such as megakaryocytes, platelets, CD34 + cells and primitive progenitor cells (Debili, N et al., Blood, 85: 391 -401 (1995); de Sauvage, FJ et al, Nature, 369: 533-538 (1994); Bartley, TD et al., cell, 77:. 1117-1124 (1994); Lok, S et al, Nature, 369: 565 8 (1994)). 与白细胞介素和蛋白质激素的绝大多数受体类似,c-Mpl属于I类细胞因子超家族(Vigon,I.等,美国科学院院刊,89:5640-5644(1992))。 And interleukins and protein hormones vast majority of similar receptors, c-Mpl belongs to the class I cytokine superfamily (Vigon, I et al., Proc Natl Acad Sci USA, 89: 5640-5644 (1992)). 这类受体的激活包括配体结合诱导的受体同二聚体化,并依次引发一系列的信号转导事件。 Such activating receptors include ligand binding induced receptor homodimerization, and in turn trigger a series of signaling events.

一般说来,一种蛋白质配体与其受体的相互作用通常发生在一个相对较大的界面。 Generally, the interaction of a protein ligand with its receptor usually occurs in a relatively large interface. 但是,人生长激素与其受体结合的例证却显示,界面上只有几个关键基团真正参与了绝大部分的结合活动(Clackson,T.等,科学,267:383-386(1995))。 However, examples of human growth hormone to its receptor have shown, the interface only a few really key groups involved in most of the binding activity (Clackson, T et al., Science, 267: 383-386 (1995)). 这一点和大量剩余蛋白质配体仅仅起到在正确的拓扑结构下显示结合表位的事实,使寻找小型活性配体成为可能。 This large amount of the remaining protein ligand and functions only in the fact that the correct topology display epitope binding the active ligand to find small as possible.

一种向此方向迈进的努力,噬菌体肽文库显示系统作为一种鉴定大型蛋白配体的模拟小型肽的强有力技术,业已浮出(Scott,JK等,科学,249:386(1990);Devlin,JJ等,科学,249:404(1990))。 Moving one kind of the efforts in this direction, the phage peptide library display system as a powerful technology to simulate large small peptide ligand identification, has emerged (Scott, JK et al., Science, 249: 386 (1990); Devlin , JJ et al., Science, 249: 404 (1990)). 通过应用这种技术,发现了作为c-Mpl受体激动剂的小型肽分子(Cwirla,SE等,科学,276:1696-1699(1997))。 By using this technique, small peptide molecules discovered (Cwirla, SE et al., Science, 276: 1696-1699 (1997)) as the c-Mpl receptor agonist.

在这样一项研究中,作为与丝状噬菌体衣壳蛋白融合的随机小型肽序列用抗体固定化的c-Mpl细胞外功能域亲和洗脱,剩余的噬菌体经第二轮亲和纯化富集。 In such a study, random small peptide sequences as fused to filamentous phage coat protein antibodies immobilized c-Mpl extracellular domain affinity and eluted phage remaining after the second round of affinity enrichment and purification . 这一结合选择和再增殖过程重复多次,以增加紧密结合者的库。 This binding selection and repopulation process is repeated several times to increase the library's close connection. 这样,首次鉴定了两个c-Mpl结合肽家族,其序列彼此不相关。 Thus, the first two identified family of c-Mpl-binding peptides, unrelated to each other in the sequence. 然后创建诱变文库以进一步优选最佳结合者,最终分离获得一种极高活性肽,其IC50=2nM,EC50=400nM(Cwirla,SE等,科学,276:1696-1699(1997))。 To further mutagenesis library then create the best binders preferably, the final separation to obtain a high active peptide which IC50 = 2nM, EC50 = 400nM (Cwirla, SE et al., Science, 276: 1696-1699 (1997)). 这一命名为TMP(TPO模拟肽)的含有14个基团的肽,与TPO或MGDF没有明显的序列同源性。 This peptide contains 14 named group is the TMP (TPO mimetic peptide), and to TPO or MGDF no significant sequence homology. 这一TMP化合物的结构如下:Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala序列1或IEGPTLRQWLAARA以氨基酸缩写的单个字母表示。 The structure of this TMP compound is as follows: Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala 1 or IEGPTLRQWLAARA sequence represented by single letter amino acid abbreviations.

以前,在一项关于EPO模拟肽的相似研究中,应用相同的技术发现了EPO模拟肽(EMP)(Wrighton,NC等,科学,273:458-463(1996)),并发现其以二聚体的形式与EPO受体(EPOR)结合。 Previously, in a similar study on EPO mimetic peptides, the same application technology found EPO mimetic peptide (EMP) (Wrighton, NC et al., Science, 273: 458-463 (1996)), and found to be dimeric form of the EPO receptor body (the EPOR) binding. 根据X线晶体照相术资料,形成的(配体)2/(受体)2复合物C2对称(Livnah,O.等,科学,273:464-471(1996))。 The X-ray crystallography data is formed (ligand) 2 / (receptor) 2 complex C2 symmetry (Livnah, O et al., Science, 273: 464-471 (1996)). 基于这一结构资料,一种共价结合的EMP二聚体设计产生,该二聚体是将两个EMP单体的C末端与一个可变间隔区交联,并发现该二聚体无论体内/体外,结合生物活性均大大加强(Wrighton,NC等,自然生物技术,15:1261-1265(1997))。 Based on this data structure A covalently bound dimer of EMP generation design, the C-terminus of the dimer is two EMP monomers crosslinked with a variable spacer region and found that regardless of the dimer in vivo / in vitro, the biological activity was greatly enhanced binding (Wrighton, NC et al, Nature biotechnology, 15: 1261-1265 (1997)).

一种相似的C末端二聚体化策略被用于TPO模拟肽(TMP)(Cwirla,SE等,科学,276:1696-1699(1997))。 A similar C-terminal dimerization strategy was used for the TPO mimetic peptide (TMP) (Cwirla, SE et al., Science, 276: 1696-1699 (1997)). 结果发现,在细胞增殖试验中,TMP的C末端连接的二聚体(CC连接)结合亲和力改善,为0.5nM,体外活性显著增强(EC50=0.1nM)(Cwirla,SE等,科学,276:1696-1699(1997))。 It was found that cell proliferation assay, the C-terminus of TMP dimer connection (CC connection) to improve the binding affinity, as of 0.5 nM, was significantly enhanced in vitro activity (EC50 = 0.1nM) (Cwirla, SE et al., Science, 276: 1696-1699 (1997)). 该TMP CC二聚体的结构显示如下: This structure TMP CC dimer is shown below: (序列2)本发明的另一方面,该串联二聚体可以进一步与来自免疫球蛋白的一个或更多部分连接,通常是指免疫球蛋白的Fc段。 (Sequence 2) is another aspect of the present invention, the tandem dimers may be further connected to one or more parts from an immunoglobulin, typically it refers to an immunoglobulin Fc fragment. 获得的化合物称之为Fc融合的TMP串联二聚体。 Fc fusion compound obtained is called TMP tandem dimer.

下面是有关用于与某些本化合物连接的抗体Fc段的简要背景部分。 The following is a brief background section relating to the Fc is connected to a portion of certain of the present compounds.

抗体包括两个独立的功能部分,一个是被称之为“Fab”的可变区,与抗原结合,以及另一个被称之为“Fc”的稳定区,提供与效应器功能如补体固定或吞噬作用的连接。 Antibodies comprise two separate functional parts, one is called "Fab" variable regions, bound antigen, and the other is called "Fc" region of stable, provides effector functions such as complement fixation or connection phagocytosis. 免疫球蛋白的Fc部分血浆半衰期长,而Fab则短。 Fc portion of an immunoglobulin long plasma half-life, whereas the Fab is short.

业已有多种应用Fc段构建的治疗性蛋白产品,期望能够提供较长的半衰期,或整合一些存在于免疫球蛋白Fc段的功能,如与Fc受体结合,与蛋白A结合,补体固定,以及胎盘转移(Capon等,自然,337:525-531(1989))。 There have been many applications of therapeutic protein products of the Fc construct, it is desirable to provide a longer half-life, or integrated in the presence of some functional immunoglobulin Fc region, such as Fc receptor binding, binding to protein A, complement fixation, and placental transfer (the Capon et al, Nature, 337: 525-531 (1989)). 例如,IgG1抗体的Fc段业已与CD30-L融合,后者是一种可以与CD30受体结合的分子,该受体在霍奇金病肿瘤细胞、退行性淋巴瘤细胞、T细胞白血病细胞和其他恶性细胞类型表面均有表达。 For example, the Fc segment IgG1 antibody has been fused to CD30-L, which can be combined with a CD30 receptor molecules, the receptor of tumor cells in Hodgkin's disease gold, anaplastic lymphoma cells, T-cell leukemia cells and other types of malignant cell surface expression of both. 见美国专利号5 480 981。 See US Patent No. 5,480,981. 为了延长其短暂的循环半衰期,IL-10,一种抗炎抗排斥制剂,也已与鼠Fcγ2a融合(Zheng,X.等,免疫学杂志,154:5590-5600(1995))。 In order to extend its short circulating half-life, IL-10, an anti-inflammatory anti-rejection agents, has also been fused with mouse Fcγ2a (Zheng, X et al., J. Immunol. 154: 5590-5600 (1995)). 研究还评价了将肿瘤坏死因子受体与人IgG1的Fc段连接,治疗感染性休克患者的应用(Fisher,C.等,新英格兰医学杂志,334:1697-1702(1996);Van Zee,K.等,免疫学杂志,156:2221-2230(1996))。 The study also evaluated the application to connect with tumor necrosis factor receptor Fc portion of human IgG1, the treatment of patients with septic shock (Fisher, C, etc., the New England Journal of Medicine, 334:. 1697-1702 (1996); Van Zee, K et al., J. Immunol. 156: 2221-2230 (1996)). Fc段还与CD4受体融合,产生的治疗蛋白被用于艾滋病的治疗。 Fc fragment is also fused with CD4 receptor to produce a therapeutic protein is used for treatment of AIDS. 见Capon等,自然,337:525-531(1989)。 See Capon et al., Nature, 337: 525-531 (1989). 此外,白细胞介素2业已与IgG1或IgG3的Fc段融合,以克服白细胞介素2的短暂半衰期及其系统毒性。 In addition, interleukin 2 has the IgG1 or IgG3 Fc region fused to overcome the short half life of interleukin 2 and its systemic toxicity. 见Harvill等,免疫技术,1∶95-105(1995)。 See Harvill et al., Immunological techniques, 1:95-105 (1995).

尽管已经可以通过商业途径得到TPO和MGDF,但提供具有刺激血小板产生(血小板生成活性)和/或血小板前体细胞,特别是巨核细胞产生(巨核细胞生成活性)的生物活性的其他化合物,仍然存在需求。 Although it has been TPO and MGDF commercially, but to provide a stimulating platelet production of other compounds (thrombopoietic activity) and / or platelet precursor cells, especially megakaryocytes to produce biologically active (megakaryocytopoiesis activity) still exists demand. 本发明提供具有这些活性及相关方面的新型化合物。 The present invention provides novel compounds having these activities and related aspects.

发明概述本发明提供一组化合物,这些化合物可以结合并通过,即激活c-Mpl受体引发跨膜信号,所述c-Mpl受体与介导内源性血小板生成素(TPO)的受体相同。 SUMMARY The present invention provides a group of compounds which may be bonded by, i.e., activation of c-Mpl receptor induced transmembrane signaling, the c-Mpl receptor mediating endogenous thrombopoietin (TPO) receptor the same. 因此,本发明的化合物具有血小板生成活性,即在体内外刺激血小板产生的能力,和/或巨核细胞生成活性,即在体内外刺激血小板前体产生的能力。 Accordingly, the compounds of the present invention have the ability thrombopoietic activity, i.e., stimulation of platelet production in vivo, and / or megakaryocyte formation activity, i.e. ability to stimulate platelets before the body is produced in vivo.

在第一个优选实施方案中,本发明化合物包括以下常见结构:TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下核心结构的一组化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4选自脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸; In a first preferred embodiment, the compounds of the present invention include the following common structure: TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group of compounds comprising the core structure: X2-X3-X4-X5- X6-X7-X8-X9-X10 wherein, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is selected from proline; X5 is selected from threonine and serine; X6 is selected from leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine, tianmen asparagine and glutamic acid; X9 is selected from tryptophan, tyrosine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine, and lysine;

L1为本文所述接头;并且n是0或1;及其生理学可以接受的盐类。 L1 is a linker as described herein; and n is 0 or 1; and their physiologically acceptable salts thereof.

在一个实施方案中,L1包括(甘氨酸)n,其中n为1-20,并且当n大于1时,高达一半的甘氨酸残基可以用选自其他19种天然氨基酸或其立体异构体的氨基酸所取代。 In one embodiment, Ll comprises (glycine) n, where n is 1-20, and when n is greater than 1, up to half of the glycine residue may be selected from the other 19 natural amino acids or a stereoisomer thereof replaced.

TMP1和TMP2的核心结构除了上述公开的X2-X10外,还有其他相关结构的可能,其中一个或多个下述基团可以添加到TMP1和/或TMP2的核心结构上:X1被连接到N末端和/或X11,X12,X13,和/或X14被连接到C末端,其中X1,X12,X13和X14如下所述:X1选自异亮氨酸、丙氨酸、缬氨酸、亮氨酸、丝氨酸和精氨酸;X11选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、赖氨酸、组氨酸和谷氨酸;X12选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、甘氨酸、丝氨酸和谷氨酰胺;X13选自精氨酸、赖氨酸、苏氨酸、缬氨酸、天门冬酰胺、和甘氨酸;并且X14选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、苏氨酸、精氨酸、谷氨酸和甘氨酸。 TMP1 and TMP2 core structure disclosed in addition to the X2-X10, other structures may be associated, wherein the one or more of the following groups may be added to the core structure TMP1 and / or TMP2 is: X1 is connected to the N terminus and / or X11, X12, X13, and / or X14 are connected to the C-terminus, wherein X1, X12, X13 and X14 follows: X1 is selected from isoleucine, alanine, valine, leucine acid, serine, and arginine; X11 is selected from alanine, isoleucine, valine, leucine, phenylalanine, serine, threonine, lysine, histidine and glutamic acid ; X12 is selected from alanine, isoleucine, valine, leucine, phenylalanine, glycine, serine and glutamine; X13 is selected from arginine, lysine, threonine, valine, threonine, asparagine, and glycine; and X14 is selected from alanine, isoleucine, valine, leucine, phenylalanine, threonine, arginine, glutamic acid and glycine.

在第二个优选实施方案中,本发明化合物具有以下通式:(Fc)m-(L2)q-TMP1-(L1)n-TMP2-(L3)r-(Fc)p其中TMP1,TMP2和n分别与上述相同;L1,L2和L3是分别独立选自这里描述的接头基团的接头基团;Fc是免疫球蛋白(将在下面给出定义)的Fc段;m,p,q和r是分别独立选自0和1的数字,其中m或p至少有一个是1,而且,如果m为0则q为0,如果p为0则r为0;及其生理学可以接受的盐类。 In a second preferred embodiment, the compounds of the present invention have the general formula: (Fc) m- (L2) q-TMP1- (L1) n-TMP2- (L3) r- (Fc) p wherein TMP1, TMP2 and n are same as described above; L1, L2 and L3 are linker linker group are independently selected from the group described herein; immunoglobulin Fc (to be defined in the following) of the Fc; m, p, q, and r is 0 and each independently selected from the numbers 1, wherein at least one of m or p is 1, and, if m is 0 then q is 0, p is 0 if r is 0; and physiologically acceptable salts thereof . 在一个实施方案中,L1,L2和L3独立包括(甘氨酸)n,其中n为1-20,并且当n大于1时,高达一半的甘氨酸残基可以用选自其他19种天然氨基酸或其立体异构体的氨基酸所取代。 In one embodiment, L1, L2 and L3 independently comprise (Gly) n, where n is 1-20, and when n is greater than 1, up to half of the glycine residue may be selected from the other 19 natural amino acids or a stereoisomer substituted amino acid isomers.

上述化合物(下面有详述)的衍生物也包括在本发明中。 Derivatives of the above compounds (described in detail below) are also included in the present invention.

本发明的化合物优选肽,所述肽可以通过标准合成法或其他任何肽制备法制备。 Preferred compounds of the present invention is a peptide, the peptide may be prepared by standard synthetic methods or any other peptide preparation method. 包含非肽部分的本发明化合物除了可通过标准肽化学反应外,在适宜的情况下,可通过标准有机化学反应合成。 Compounds of the invention comprises a non-peptide moiety may be by addition to standard peptide chemistry reactions outside, under appropriate circumstances, it may be synthesized by standard organic chemistry reactions.

本发明化合物可通过与适宜的药物载体物质整合,并给予目标受体如人(或其他哺乳动物)有效剂量,用于治疗或预防目的。 The compounds of this invention can be integrated with a suitable pharmaceutical carrier materials and administering target receptor such as a human (or other mammal) an effective dose for therapeutic or prophylactic purposes. 其他相关方面也包含在本发明中。 Other related aspects are also included in the present invention.

附图简述本发明的大量其他方面和优势将在考虑下面的详细描述并参考附图后变得清晰,其中:图1举例显示了人IgG1的Fc多核苷酸和蛋白序列(序列3是编码链,阅读方向为5'→3',序列4是互补链,阅读方向是3'→5';序列5是编码氨基酸的序列),该序列可用于本发明的Fc融合化合物。 Numerous other aspects and advantages of the present invention will BRIEF becomes consideration of the following detailed description with reference to the accompanying drawings and the clear, wherein: Figure 1 shows an example of the Fc polynucleotide and protein sequences of human IgG1 (the sequence encoding 3 chain, the reading direction of 5 '→ 3', 4 is the complementary strand sequence, the reading direction is 3 '→ 5'; 5 sequence is a sequence encoding an amino acid), which can be used in sequence Fc fusion compound of the present invention.

图2显示了制备聚乙二醇化肽19(序列17)的一种合成方案。 Figure 2 shows a synthetic scheme for the preparation of pegylated peptide 19 (SEQ ID 17).

图3显示了制备聚乙二醇化肽20(序列18)的一种合成方案。 Figure 3 shows a synthetic scheme for the preparation of pegylated peptide 20 (SEQ ID 18).

图4显示了给正常BDF1雌性小鼠弹丸注射一次100μg/kg化合物后体内血小板产生的计数,注射的各种化合物如下:PEG-MGDF表示将平均分子量为20kD的PEG通过还原氨基化连接于人天然TPO的1-163氨基酸的氨基基团N末端,所述TPO在大肠杆菌中表达(因此不是糖基化的)(见题为“刺激巨核细胞生长发育的组合物和方法”的WO 95/26746);TMP表示序列1的化合物;TMP-TMP表示序列21的化合物;PEG-TMP-TMP表示序列18的化合物,其中PEG基团是平均分子量为5kD的PEG如图3所示连接;TMP-TMP-Fc将在下面给出定义,Fc-TMP-TMP除了Fc基团是连接于TMP-TMP肽的N末端而非C末端外,与TMP-TMP-Fc完全一致。 Figure 4 shows the counting after a bolus injection of normal BDF1 female mice once 100μg / kg of the compound in vivo platelet production, injection of various compounds are as follows: PEG-MGDF represents an average molecular weight of 20kD PEG linked to a human natural by reductive amination N-terminal amino group of amino acids 1-163 of TPO, the TPO expressed in E. coli (thus not glycosylated) (see, entitled "compositions and methods for stimulating megakaryocyte growth and development" in WO 95/26746 ); a compound represented by the TMP sequence of; compound 21 TMP-TMP sequence represented; PEG-TMP-TMP compound represented by the sequence of 18, wherein the PEG group is a PEG of average molecular weight of 5kD 3 is connected; TMP-TMP C-terminal extracellular N-terminus and not defined in the following will be given -Fc, Fc-TMP-TMP in addition Fc group is attached to the TMP-TMP peptide, consistent with TMP-TMP-Fc.

图5显示了通过埋植渗透泵连续7天给予正常BDF1雌性小鼠各种化合物后体内血小板产生的计数。 Figure 5 shows the implanted osmotic pumps counted by 7 consecutive days of normal female BDF1 mice platelets after various compounds. 化合物的定义与上述图4给出的完全一致。 Fully consistent with the above definition of compounds 4 are given in FIG.

图6A,6B和6C显示了本发明优选化合物的设计图。 6A, 6B, and 6C show the design of the preferred compounds of the present invention. 图6A显示了一种Fc融合化合物,其中Fc部分融合于TMP二聚体的N末端,而且Fc部分是单体(单链)形式。 6A shows one kind of N-terminal Fc fusion compound wherein the Fc portion is fused to the TMP dimer, and the Fc portion is a monomeric (single chain) form. 图6B显示了一种Fc融合化合物,其中Fc段融合于TMP二聚体的N末端,而且Fc部分为二聚体,其中一个Fc单体连接于TMP二聚体。 6B shows an Fc fusion compound wherein TMP dimers fused to the N-terminus of the Fc, and the Fc portion is a dimer, wherein one Fc monomer is attached to the TMP dimer. 图6C显示了一种Fc融合化合物,其中Fc部分与TMP二聚体的N末端连接,而且Fc部分为二聚体,每一Fc单体与一个TMP二聚体连接。 6C shows an Fc fusion compound wherein the Fc portion is connected to the N-terminus of the TMP dimer, and the Fc portion is dimeric, each Fc monomer and a TMP dimer connected.

优选实施方案详述在寻求小型结构作为引导化合物以发展拥有更多期望特征的治疗性制剂的努力下,一种不同类型的TMP二聚体及相关结构设计出台,其中一个TMP肽的C末端直接或通过一个接头与第二个TMP肽的N末端相连,并随之评价这种二聚体策略对获得的二聚体分子的生物活性的影响。 DESCRIPTION OF PREFERRED EMBODIMENTS In efforts to seek small structures as a guide to the development of compounds with more desirable characteristics of the therapeutic agent, a different type of dimer of TMP and related structures design introduced in which a C-terminal peptide is directly TMP or the N-terminus by a linker connected to a second TMP peptide, and subsequently evaluate the effect of this strategy on a dimer of the biologically active dimeric molecules obtained. 在一些情况下,这些被称之为串联的二聚体(CN连接)在两个单体之间被设计加入接头,所述接头优选包括天然氨基酸,因此其合成可以应用重组技术。 In some cases, these are called tandem dimers (CN connection) between the two monomers are added to design a joint that preferably comprises a natural amino acid, so it can be synthesized using recombinant techniques.

本发明基于一组具有血小板生成活性的化合物的发现,这些化合物通过与内源性TPO受体c-Mpl连接以发挥作用。 The present invention generates a discovery of the active compound based on a set having a platelet, which is connected with an endogenous compound c-Mpl TPO receptor to function.

化合物及衍生物在第一个优选实施方案中,本发明化合物包括以下一般结构:TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下核心结构的一组化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4选自脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为本文所述接头;并且n是0或1;及其生理学可以接受的盐类。 Compounds and derivatives In a first preferred embodiment, the compounds of the present invention comprises the following general structure: TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group of compounds comprising the core structure: X2-X3- X4-X5-X6-X7-X8-X9-X10 wherein, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is selected from proline ; X5 is selected from threonine and serine; X6 is selected from leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from Valley ammonia amides, asparagine and glutamic acid; X9 is selected from tryptophan, tyrosine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine acid, methionine and lysine; Ll is a linker as described herein; and n is 0 or 1; and their physiologically acceptable salts thereof.

在一个实施方案中,L1包括(甘氨酸)n,其中n为1-20,并且当n大于1时,高达一半的甘氨酸残基可以用选自其他19种天然氨基酸或其立体异构体的氨基酸所取代。 In one embodiment, Ll comprises (glycine) n, where n is 1-20, and when n is greater than 1, up to half of the glycine residue may be selected from the other 19 natural amino acids or a stereoisomer thereof replaced.

TMP1和TMP2的核心结构除了上述公开的X2-X10外,还有其他相关结构的可能,其中一个或多个下述基团可以添加到TMP1和/或TMP2的核心结构上:X1被连接到N末端和/或X11,X12,X13,和/或X14被连接到C末端,其中X1,X11,X12,X13和X14如下所述:X1选自异亮氨酸、丙氨酸、缬氨酸、亮氨酸、丝氨酸和精氨酸;X11选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、赖氨酸、组氨酸和谷氨酸;X12选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、甘氨酸、丝氨酸和谷氨酰胺;X13选自精氨酸、赖氨酸、苏氨酸、缬氨酸、天门冬酰胺、和甘氨酸;并且X14选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、苏氨酸、精氨酸、谷氨酸和甘氨酸。 TMP1 and TMP2 core structure disclosed in addition to the X2-X10, other structures may be associated, wherein the one or more of the following groups may be added to the core structure TMP1 and / or TMP2 is: X1 is connected to the N terminus and / or X11, X12, X13, and / or X14 are connected to the C-terminus, wherein X1, X11, X12, X13 and X14 follows: X1 is selected from isoleucine, alanine, valine, leucine, serine and arginine; X11 is selected from alanine, isoleucine, valine, leucine, phenylalanine, serine, threonine, lysine, histidine and valleys acid; X12 is selected from alanine, isoleucine, valine, leucine, phenylalanine, glycine, serine and glutamine; X13 is selected from arginine, lysine, threonine , valine, asparagine, and glycine; and X14 is selected from alanine, isoleucine, valine, leucine, phenylalanine, threonine, arginine, glutamic acid, and glycine.

术语“TMP”用来表示由,即包括至少9个亚单位(X2-X10)组成的部分,其中X2-X10组成核心结构。 The term "TMP" is used to indicate a, i.e., comprising at least 9 subunits (X2-X10) composed of a portion, which formed the core structure X2-X10. X2-X14的亚单位优选独立选自20个天然氨基酸的氨基酸,但是,本发明也包括X2-X14独立选自本领域熟知的非典型、非天然氨基酸的化合物。 X2-X14 subunits are preferably amino acids independently selected from the 20 natural amino acids, however, the present invention also includes atypical known in the art, unnatural amino compound X2-X14 are independently selected. 每一位置特别优选的氨基酸已经鉴定。 Particularly preferred amino acid at each position has been identified. 例如,X2可以是谷氨酸,天门冬氨酸,赖氨酸或缬氨酸。 For example, an X2 may be glutamic acid, aspartic acid, lysine or valine. 三个字母和单个字母的氨基酸缩写形式这里均予沿用;每种情况下,缩写都是20种天然氨基酸或大家熟知的其变异物的标准缩写形式。 Three-letter and single letter amino acid abbreviations follow herein are herein; each case, the abbreviations are the standard 20 naturally occurring amino acids or well-known in its abbreviated form variants thereof. 这些氨基酸在立体化学上可以是L型或D型(除了甘氨酸,既没有L型,也没有D型),TMPs可以包括立体化学的组合。 These amino acids may be stereochemically L or D form (except glycine, neither L-type, D-type nor), TMPs may comprise a combination of stereochemistry. 但在TMP链中优选所有氨基酸的立体化学L型。 However, in the TMP chain stereochemistry of all amino acids is preferably the L form. 本发明还提供反向TMP分子,其中羧基末端的氨基酸序列是反向的。 The present invention also provides reverse TMP molecules wherein the amino acid sequence of the carboxy terminus is reversed. 例如,具有正常序列X1-X2-X3分子的反向序列为X3-X2-X1。 For example, the reverse sequence having the normal sequence X1-X2-X3 molecule is X3-X2-X1. 本发明还提供逆向-反向TMP分子,其中,与反向TMP一样,羧基末端的氨基酸序列是反向的,而且TMP中通常情况下是“L”镜像体的残基改变为“D”立体异构体形式。 The present invention further provides a reverse - reverse TMP molecules wherein, like a reverse TMP, the amino acid sequence of the carboxy terminus is reversed, and is usually in the TMP "L" residue changes the mirror body is "D" perspective isomeric forms.

此外,TMPs的生理学可以接受的盐类也包括在内。 In addition, TMPs physiologically acceptable salts are also included. “生理学可以接受的盐类”表示任何已知或后来发现的可以药用的盐类。 "Physiologically acceptable salt" means any pharmaceutically acceptable salt known or later discovered. 一些特别优选的实例如:乙酸盐、三氟乙酸盐、盐酸盐、氢溴酸盐、硫酸盐、枸橼酸盐、酒石酸盐、羟乙酸盐、草酸盐。 Some particularly preferred examples such as: acetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, oxalate.

还可考虑应用TMPs的“衍生物”来取代上述TMPs。 TMPs may also consider the application "derivative" instead of the above-described TMPs. 这些TMPs的衍生物包括经一个或多个下述方法修饰的部分:一个或多个肽[-C(O)NR-]连接(键)被非肽连接如-CH2-氨甲酸酯连接[-CH2-OC(O)NR-]所取代;一个膦酸酯连接;一个-CH2-磺胺[-CH2-S(O)2NR-]连接;一个脲[-NHC(O)NH-]连接;一个-CH2-仲胺连接;或者一个烷基化的肽连接[-C(O)NR6-其中R6是低级烷基];以下肽,其中N末端衍生为一个-NRR1基团;为一个-NRC(O)R基团;为一个-NRC(O)OR基团;为一个-NRS(O)2R基团;为一个-NHC(O)NHR基团,其中R和R1是氢原子或低级烷基,但条件是R和R1不能同时为氢原子;为一个琥珀酰亚胺基团;为一个苄氧羰基-NH-(CBZ-NH-)基团;或一个苄氧羰基-NH-基团,其中苯环有1-3个取代物,分别选自低级烷基、低级烷氧基、氯和溴;并且以下肽,其中游离C末端衍生为-C(O)R2,其中R2选自低级烷氧基,以及-NR3R4,其中R3和R4独立选自氢原子和低级烷基。 Derivative TMPs include those with one or more of the following methods modified parts: one or more peptide [-C (O) NR-] connection (bond) is connected as non-peptide connecting the urethane -CH2- [ -CH2-OC (O) NR-] substituted; a phosphonate connection; a -CH2- sulfonamide [-CH2-S (O) 2NR-] connection; a urea [-NHC (O) NH-] is connected; a secondary amine linkage -CH2-; or a connection alkylated peptide [-C (O) NR6- wherein R6 is lower alkyl]; the peptide, wherein the N-terminus derivatized to a -NRR1 group; a -NRC (O) R group; to a -NRC (O) oR group; a -NRS (O) 2R group; a -NHC (O) NHR group, wherein R and R1 is a hydrogen atom or a lower alkyl group, with the proviso that R and R1 are not simultaneously a hydrogen atom; a succinimide group; a benzyloxycarbonyl group -NH- (CBZ-NH-) group; or a benzyloxycarbonyl group -NH- , wherein the phenyl ring with 1-3 substituents, independently selected from lower alkyl, lower alkoxy, chlorine and bromine; and the following peptides wherein the free C terminus is derivatized -C (O) R2, wherein R2 is selected from lower alkoxy and -NR3R4, wherein R3 and R4 are independently selected from hydrogen atoms and lower alkyl. “低级”的意思是含有1-6个碳原子的基团。 "Lower" means groups containing 1 to 6 carbon atoms.

此外,各个氨基酸也可修饰后引入TMP分子,方法是通过将肽的靶向氨基酸残基与一种有机衍生剂反应,该衍生剂能够与所选支链和末端残基发生反应。 Further, each of the amino acids may also be introduced into the TMP molecule modified, by reacting targeted amino acid residues of the peptide with an organic derivatizing agent capable of reaction with the derivatizing agent is selected branched chain and terminal residue. 下面是实例:赖氨酸和氨基末端残基可以与琥珀酸或其他羧酸酐反应。 The following are examples: lysine and amino terminal residues may be reacted with succinic anhydride or other dicarboxylic acids. 用这些试剂进行衍生反应的效果就是可以逆转赖氨酸残基的电荷。 Effect derivatization reactions can be reversed with these agents is charged lysine residues. 其他可以使含有α-氨基的残基发生衍生反应的适宜试剂包括:亚氨酸酯如甲基甲基砒啶亚氨酸酯(methyl picolinimidate);磷酸吡哆醛;吡哆醛;氯硼氢化物(chloroborohydr ide);三硝基苯磺酸;O-甲基异脲;2,4戊二酮;和转氨酶催化的与乙醛酸发生的反应。 Other suitable agent residue containing α- amino group may occur derivatization reactions include: imidate esters such as methyl imidate lutidine (methyl picolinimidate); pyridoxal phosphate; pyridoxal; chloro borohydride was (chloroborohydr ide); trinitrobenzene sulfonic acid; 0- methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate occurs.

精氨酸残基可以通过与一个或几个常规试剂发生反应得到修饰,所述试剂包括苯乙醛、2,3-丁二酮,1,2-环己二酮,和茚三酮。 Arginine residues can be modified by reaction with one or several conventional reagents, the reagent comprises phenylacetaldehyde, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. 精氨酸残基的衍生反应需要在碱性条件下进行,因为胍官能团的pKa很高。 The reaction of arginine residues derived needs to be performed under alkaline conditions because of the high pKa of the guanidine functional group. 而且,这些试剂可与赖氨酸和精氨酸的胍基团均发生反应。 Furthermore, these reagents may lysine and arginine guanidine group are reacted.

酪氨酸残基的特异性修饰业已经过深入研究,特别引人关注的是通过与芳族重氮化合物或四硝基甲烷反应,将光谱标记引入酪氨酸残基。 The specific modification of tyrosine residues have been intensively studied, with particular interest by aromatic diazonium compounds or tetranitromethane reaction, introducing spectral labels into tyrosine residues. 最常见的是,氮-乙酰咪唑和四硝基甲烷可被分别用来构建氧-乙酰酪氨酸类和3-硝基衍生物。 Most commonly, N - acetyl imidazole and tetranitromethane may be used to construct separately oxo - acetyl tyrosine species and 3-nitro derivatives.

羧基侧基(天门冬氨酸或谷氨酸)可以通过与碳二亚胺(R'-N=G=N-R')如1-环乙基-3-(2-吗啉基-(4-乙基)碳二亚胺或1-乙基-3-(4-氮鎓-4,4-二甲基戊基)碳二亚胺反应而被选择性修饰。进一步,天门冬氨酸和谷氨酸残基还可通过与铵离子反应转变为天门冬酰胺和谷氨酰胺残基。 Carboxyl side groups (aspartic acid or glutamic acid) by reaction with carbodiimides (R'-N = G = N-R ') such as 1-ethyl-3- (2-morpholin-yl - ( 4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide be selectively modified. further, aspartate and it may also be converted to glutamic acid residues asparagine and glutamine residues by reaction with ammonium ions.

谷氨酰胺和天门冬酰胺残基经常脱氨基转变为相应的谷氨酸和天门冬氨酸残基。 Glutamine and asparagine residues are often converted into the corresponding deaminated to glutamic acid and aspartic acid residues. 或者,这些残基可在微酸性条件下脱氨基。 Alternatively, these residues may be deaminated under mildly acidic conditions. 这些残基的任一形式都包括在本发明范围内。 Either form of these residues are included within the scope of the present invention.

用双功能试剂衍生对于将肽或其功能衍生物交联到不溶于水的支持基质或其他大分子载体上是有用的。 Derivatization with bifunctional agents is useful for peptide or functional derivatives thereof cross-linked to a water-insoluble support matrix or other macromolecular carriers. 常用的交联试剂包括,例如,1,1-双(重氮基乙酰)-2-苯乙烷,戊二醛,N-羟基琥珀酰亚胺酯,例如,4-叠氮水杨酸酯,同双功能的亚氨酸酯,包括二琥珀酰亚胺基酯如3,3-二硫双(琥珀酰亚胺基丙酸盐),和双功能的马来酰亚胺如双-N-马来酰亚胺-1,8-辛烷。 Commonly used crosslinking agents include, e.g., 1,1-bis (diazo-acetyl) -2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, 4-azido salicylate , homobifunctional imidoesters, including disuccinimidyl esters such as 3,3-dithio-bis (succinimidyl propionate), and bifunctional maleimides such as bis -N - maleimido-1,8-octane. 衍生试剂如甲基-3-[(对叠氮苯)二硫]丙酰亚氨酸酯(propioimidate)可以产生光激活中间物,在有光存在的情况下可以形成交联。 Derivatizing agent such as methyl-3 - [(p-azido phenyl) dithio] propionyl crosslinked imidate (propioimidate) may generate light activated intermediates may be formed in the presence of light. 此外,反应性不溶于水的基质如在美国专利号为3 969 287;3 691 016;4 195 128;4 247 642;4 229 537和4 330 440中描述的溴化氰激活的碳水化物和反应性底物可用于蛋白质固定化。 Further, reactive water-insoluble matrices such as in U.S. Patent No. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 cyanogen bromide and 4,330,440 describe the reaction of activated carbohydrates and of the substrate can be used for protein immobilization.

其他可能的修饰包括脯氨酸和赖氨酸的羟基化,丝氨酸或苏氨酸残基羟基基团的磷酸化,半胱氨酸中硫原子的氧化,赖氨酸、精氨酸和组氨酸侧链α-氨基基团的甲基化(Creighton,TE等,蛋白质:结构和分子特性,WHFreeman & Co.有限公司,旧金山,79-86页(1983)),N末端氨基的乙酰化,并且,在一些情况下,是C末端羧基基团的酰胺化。 Other possible modifications include phosphorylation hydroxylation of proline and lysine, serine or threonine residue of a hydroxyl group, the oxidation of the sulfur atom of a cysteine, lysine, arginine, and histidine α- amino acid side chains of methyl group (Creighton, TE et al., proteins: structure and molecular properties, WHFreeman & amp; Co., Ltd., San Francisco, pp. 79-86 (1983)), N-terminal acetylation of the amino group and, in some instances, amidation of the C-terminal carboxyl group.

本发明化合物的这些衍生部分优选改善一个或多个特征,包括血小板生成活性,溶解性,吸收,生物半衰期等。 These parts are preferably derived from compounds of the invention to improve one or more characteristics including thrombopoietic activity, solubility, absorption, biological half life and the like. 另外,衍生部分获得的化合物要与没有衍生的化合物具有相同,或大致相同的特征和/或特性。 Further, the obtained compound derived portion to have the same, or substantially the same features and / or characteristics of the compound is not derived. 所述部分应可根除或减弱化合物的不利副反应等。 The part should be eradicated or reduced adverse side reactions like compound.

除了上述的核心结构X2-X10,还具体考虑的其他结构包括有一个或多个X基团与核心结构相连的结构。 In addition to the core structure X2-X10, other structures are also specifically contemplated include one or more structures connected to the X group to the core structure. 因此,X1,和/或X11,X12,X13和X14可以连接到核心结构。 Thus, X1, and / or X11, X12, X13 and X14 may be connected to the core structure. 下面给出一些其他示范结构: Here are some other exemplary structures:

X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-Xl2-X13-X14,其中X1-X14如上述。 X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12; X2-X3-X4-X5- X6-X7-X8-X9-X10-X11-X12-X13; X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14; X1-X2-X3-X4- X5-X6-X7-X8-X9-X10; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; X1-X2-X3-X4-X5-X6-X7-X8- X9-X10-X11-X12; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13; X1-X2-X3-X4-X5-X6-X7-X8- X9-X10-X11-Xl2-X13-X14, wherein X1-X14 as described above. 每个TMP1和TMP2在序列和/或长度上可以一致,也可以不同。 Each TMP1 and TMP2 may be identical in sequence and / or length, may be different. 在一些优选实施方案中,TMP1和TMP2相同。 In some preferred embodiments, TMP1 and TMP2 same.

一个特别优选的TMP如下:Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala(序列1)这里所用的“包括”的意思是,除了其他事物外,一个化合物可以包含在给定序列的-或C末端同时或在任一端添加氨基酸。 A particularly preferred TMP is the following: Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala (sequence 1) As used herein "comprising" means that, among other external things, the compound may be contained in a given sequence - or C-terminus simultaneously or addition of amino acids at either end. 但是,只要如X2-X10,X1-X14,或其他示例结构存在,余下的化学结构就相对不重要了。 However, as long as X2-X10, X1-X14, other exemplary structures is present, the remaining chemical structure is relatively less important. 当然,核心结构X2-X10或X1-X14以外的任何结构,都不应显著影响化合物的血小板生成活性。 Of course, any structure outside of the core structure X2-X10 or X1-X14, should not significantly affect platelet production of the active compound. 例如,一个N末端蛋氨酸残基就被视为属于本发明。 For example, a N-terminal methionine residue to be regarded as belonging to the present invention. 此外,尽管本发明的多种优选化合物为串联二聚体,这种二聚体拥有两个TMP部分,但本发明的其他化合物为串联多聚体TMPs,即拥有下述示例结构的化合物:TMP1-L-TMP2-L-TMP3TMP1-L-TMP2-L-TMP3-L-TMP4TMP1-L-TMP2-L-TMP3-L-TMP4-L-TMP5;其中,TMP1,TMP2,TMP3,TMP4和TMP5的结构可以相同也可以不同,并且每个TMP和L如在此所定义的,每个接头都是可选的。 Compound addition, although various preferred compounds of the present invention is a tandem dimer, the dimer of TMP has two portions, but other compounds of this invention are tandem multimers TMPs, i.e., have the following exemplary structures: TMP1 -L-TMP2-L-TMP3TMP1-L-TMP2-L-TMP3-L-TMP4TMP1-L-TMP2-L-TMP3-L-TMP4-L-TMP5; wherein, TMP1, TMP2, TMP3, TMP4, and TMP5 of structure may be the same or different, and each TMP and L are as defined herein, each linker is optional. 本发明化合物优选2-5个TMP部分,特别优选2-3个,最优选2个。 Preferred compounds of the present invention, TMP portion 2-5, particularly preferably 2-3, most preferably 2. 本发明第一个实施方案的化合物优选总数少于大约60个氨基酸,更优选少于大约40个氨基酸(即,他们是肽)。 The total number of preferred compounds of the first embodiment of the present invention is less than about 60 amino acids, more preferably less than about 40 amino acids (i.e., they are peptides).

如上所述,本发明第一个实施方案的化合物优选或者直接连接或者与一个接头基团相连的TMP二聚体。 As described above, the compounds of the first embodiment of the present invention are preferably TMP dimers or directly or with a linker group is attached. 单体TMP部分的显示方式从左向右阅读是常规的从N末端到C末端的定向。 TMP monomers display section is read from left to right orientation of conventional N-terminus to C-terminus. 据此,我们可以看到,本发明化合物全部是有方向定位的,因此TMP1的C末端或者直接或者通过一个接头与TMP2的N末端相连。 Accordingly, we can see that all of the compounds of the present invention is positioning direction, and therefore the C-terminus of TMP1 or connected directly or via a linker to the N-terminus of TMP2. 这种定向被称之为串联定向,并且本发明化合物通常被称之为“串联二聚体”。 This orientation is called tandem orientation, and the present compounds are usually called "tandem dimer." 即使TMP1和TMP2的结构是不同的,这些化合物仍被称为二聚体。 TMP1 and TMP2 even if the structure is different, these compounds are still referred to as a dimer. 也就是说,这里包括同二聚体和异二聚体。 In other words, here including homodimers and heterodimers.

“接头”基团(“L1”)是可选的。 "Linker" group ( "Ll") is optional. 当其存在时,对其化学结构并无严格要求,因为它的主要作用是作为间隔区。 When present, its chemical structure is not critical, because its main role as a spacer. 接头应有所选择,以致不会影响最终化合物的生物活性,也不会显著增强最终化合物的免疫原性。 Joints shall be selected so as not to affect the biological activity of the final compound is not significantly enhance the immunogenicity of the final compound. 接头优选由肽键连接在一起的氨基酸组成。 Amino acid linker is preferably connected together by peptide bonds compositions. 因此,在优选实施方案中,接头包括Yn,其中Y是天然氨基酸或其立体异构体,“n”是1-20的任意一个数字。 Thus, in a preferred embodiment, the linker comprises Yn of, wherein Y is a natural amino acid or a stereoisomer, "n" is any of a number 1-20. 因此接头由通过肽键连接的1-20个氨基酸组成,其中氨基酸选自20种天然氨基酸。 Thus linker consisting of 1-20 amino acids linked by peptide linkages, wherein the amino acid selected from the 20 naturally occurring amino acids. 在一个更优选的实施方案中,所述1-20个氨基酸选自甘氨酸、丙氨酸、脯氨酸、天门冬酰胺、谷氨酰胺、半胱氨酸和赖氨酸。 In a more preferred embodiment, the 20 amino acids selected from glycine, alanine, proline, asparagine, glutamine, cysteine ​​and lysine. 更加优选的是,接头由空间结构没有位阻的氨基酸为主体组成,例如甘氨酸,甘氨酸-甘氨酸[(甘氨酸)2],甘氨酸-甘氨酸-甘氨酸[(甘氨酸)3],……(甘氨酸)20,丙氨酸,甘氨酸-丙氨酸,丙氨酸-甘氨酸,丙氨酸-丙氨酸,等。 More preferably, the linker is not hindered by the spatial structure of an amino acid as the main component, such as glycine, glycine - Gly [(Gly) 2], Gly - Gly - Gly [(Gly) 3], ...... (glycine) 20, alanine, glycine -, alanine - glycine Ala - alanine, and the like. 接头的其他具体实例为:(甘氨酸)3赖氨酸(甘氨酸)4(序列6);(甘氨酸)3天门冬酰胺甘氨酸丝氨酸(甘氨酸)2(序列7)(当其在能够糖基化这种位点的哺乳动物细胞系统中重组生产时,该结构提供了一个糖基化位点);(甘氨酸)3半胱氨酸(甘氨酸)4(序列8);以及甘氨酸脯氨酸天门冬酰胺甘氨酸(序列9)。 Other specific examples of linkers are :( Gly) 3 Lys (Gly) 4 (SEQ ID 6); (Gly) 3 Ser asparagine glycine (Gly) 2 (SEQ ID 7) (when it is capable of glycosylation at this mammalian cell systems produced recombinantly site, the structure provides a glycosylation site); (Gly) 3 Cys (Gly) 4 (SEQ ID 8); proline and glycine asparagine glycine (SEQ ID 9).

为了解释上述命名系统,举例来说,(甘氨酸)3赖氨酸(甘氨酸)4的意思是甘氨酸-甘氨酸-甘氨酸-赖氨酸-甘氨酸-甘氨酸-甘氨酸-甘氨酸。 To explain the above nomenclature, for example, (Gly) 3 Lys (Gly) 4 means is glycine - Gly - Gly - Lys - Gly - Gly - Gly - glycine. 还优选甘氨酸和丙氨酸的组合。 Further preferably a combination of glycine and alanine.

也可应用非肽接头。 Non-peptide linker may also be applied. 例如,烷基接头如-HN-(CH2)s-CO-,其中s=2-20。 For example, alkyl linkers such as -HN- (CH2) s-CO-, wherein s = 2-20. 这些烷基接头还可进一步被任意非空间结构位阻的基团如低级烷基(如C1-C6),低级醛基,卤素(如氯,溴),氰,氨基,苯基,等取代。 These alkyl linkers may further be any non-hindered spatial structure coordinating groups such as lower alkyl (e.g. C1-C6), lower aldehyde group, a halogen (e.g. chlorine, bromine), cyano, amino, phenyl, substituted and the like.

另一种非肽接头为聚乙二醇基团,如-HN-CH2-CH2-(O-CH2-CH2)nO-CH2-CO-其中,n的取值使接头总分子量范围大约是101-5000,优选101-500。 Another non-peptide linker is a polyethylene glycol group, such as a -HN-CH2-CH2- (O-CH2-CH2) nO-CH2-CO- wherein, n for the joint value of the total molecular weight range of about 101 5000, preferably 101-500.

在本发明第一个实施方案的血小板生成化合物中,业已发现,接头的长度一般优选大约0-14个亚基(如氨基酸)。 Thrombopoietin compound in the first embodiment of the present invention, it has been found that the linker length is generally preferably about 0-14 subunits (e.g., amino acids).

肽接头可以改变,以构建与上述TMPs相同方式的衍生物。 Peptide linker can be varied to construct derivative TMPs same manner as described above.

第一组化合物还可进一步是线形或环状的。 The first group of compounds may further be linear or cyclic. “环状”意味着分子至少有两个分离,即不连续的部分彼此连接。 "Cyclic" means having at least two separate elements, i.e., discrete sections connected to one another. 例如,分子的氨基末端和羧基末端可以共价连接以形成一个环状分子。 For example, the amino terminus and carboxy terminus of the molecule can be covalently linked to form a cyclic molecule. 另外,分子可能含有两个或多个半胱氨酸残基(如,在接头中),这些残基可以通过形成二硫键而环化。 Additionally, molecules may contain two or more cysteine ​​residues (e.g., in the linker), these residues can be cyclized via disulfide bond formation. 进一步予以考虑的是,一个以上的串联肽二聚体可以连接以形成二聚体的二聚体。 It is further contemplated that more than one tandem peptide dimer can be linked to form a dimer of dimers. 例如,一个含有半胱氨酸的串联二聚体可以与另一个这样的二聚体中的半胱氨酸形成分子内二硫键。 For example, a tandem dimer containing cysteine ​​can form an intramolecular disulfide bond with another such dimer cysteine. 见,例如下面给出的序列20的化合物。 See, for example, compound 20 of the sequence given below.

本发明化合物还可与载体分子共价或非共价连接,如一个线形聚合物(例如,聚乙二醇,聚赖氨酸,葡聚糖等),支链聚合物(见,例如1981年9月15日授予Denkenwalter等的美国专利4 289 872;1993年7月20日授予Tam的美国专利5 229 490;1993年10月28日公开的Frechet等的WO93/21259);脂质;胆固醇基团(如类固醇);或者碳水化合物或寡糖。 Compounds of the invention may also be the carrier molecule is covalently or non-covalently, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), branched-chain polymer (see, for example, 1981 US Patent September 15 granted Denkenwalter other 4,289,872; US Pat. July 20, 1993 awarded Tam's 5,229,490; October 28, 1993 open Frechet, etc. WO93 / 21259); lipids; cholesteryl groups (such as steroids); or a carbohydrate or oligosaccharide. 其他可能的载体包括一个或多个水溶性聚合物接头,如美国专利4 640 835,4 496 689,4 301 144,4 670 417,4 791192和4 179 337所述的聚乙二醇,或聚丙二醇。 Other possible carriers include one or more water-soluble polymer joint as 4640 496 689,4 835,4 301 144,4 670 417,4 791 192 and 4 179 337 according to U.S. Patent No. polyethylene glycol, or poly propylene glycol. 以及本领域熟知的其他有用聚合物,包括单甲氧基-聚乙二醇,葡聚糖,纤维素,或其他基于碳水化合物的聚合物,聚-(N-乙烯吡咯烷)-聚乙二醇,丙二醇均聚物,聚丙烯氧化物/乙烯氧化物共聚物,聚氧乙烯的多羟基化合物(如甘油)和聚乙烯醇,以及这些聚合物的混合物。 And other useful polymers known in the art, including mono-methoxy - polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly - (N-vinyl pyrrolidine) - polyethylene alcohol, propylene glycol homopolymers, a polypropylene oxide polyol / ethylene oxide copolymer, polyoxyethylene (e.g., glycerol) and polyvinyl alcohol, and mixtures of these polymers.

一种优选载体为聚乙二醇(PEG)。 A preferred carrier is polyethylene glycol (PEG). 该PEG基团可以是任意方便的分子量,可以是直链,也可以有支链。 The PEG group may be of any convenient molecular weight and may be linear, may be branched. 优选的PEG平均分子量范围为大约2kDa-100kDa,更优选5kDa-50kDa,最优选5kDa-10kDa。 The preferred average molecular weight range of PEG of about 2kDa-100kDa, more preferably 5kDa-50kDa, and most preferably 5kDa-10kDa.

PEG基团通常通过酰化作用、还原烷基化,Michael添加、硫羟烷基化或其他化学选择结合/连接方法与本发明化合物相连,通过将PEG部分的反应基团(例如,醛基、氨基、酯、巯基、α-卤代乙酰,马来酰亚胺或肼基)与靶化合物的反应基团(如,醛基、氨基、酯、巯基、α-盐乙酰,马来酰亚胺或肼基)连接达到。 PEG groups typically by acylation, reductive alkylation, Michael additions, thiol alkylation or other chemoselective binding / connection method coupled with a compound of the present invention, the reactive group of the PEG moiety (e.g., an aldehyde, amino, ester, thiol groups, alpha] haloacetyl, maleimido or hydrazino group) to a reactive group of the target compound (e.g., an aldehyde, amino, ester, mercapto, salts of alpha] acetyl maleimide or hydrazino group) to achieve the connection.

碳水化合物(寡糖)基团可以方便地连接到公认的蛋白质糖基化位点。 Carbohydrate (oligosaccharide) groups may conveniently be connected to the recognized protein glycosylation sites. 通常,当丝氨酸/苏氨酸和天门冬酰胺的排列顺序如天门冬酰胺-X-丝氨酸/苏氨酸时,O-连接的寡糖可与丝氨酸或苏氨酸残基相连,而N-连接的寡糖则与天门冬酰胺残基相连,其中,X可以是除了脯氨酸以外的任意氨基酸。 Typically, when the order of serine / threonine and asparagine, such as asparagine -X- serine / threonine, the O- linked oligosaccharides may be serine or threonine residues linked, while N- linked the oligosaccharides and asparagine residues with wherein, X may be any amino acid except proline. X优选不包括脯氨酸在内的19种天然氨基酸中的一种。 19 kinds of natural amino acids one kind of X is preferably not included in the inner proline. 发现的每种类型的N-连接和O-连接寡糖以及糖残基结构都是不同的。 Each type of N- and O- linked oligosaccharides and the sugar residues found in the structure is different. 一种通常在两者都能发现的糖类型为N-乙酰神经氨酸(即唾液酸)。 A common type of sugar can be found in both is N- acetylneuraminic acid (i.e., sialic acid). 唾液酸通常是N-连接和O-连接寡糖的末端残基,并且由于其携带负电荷的特性,可以赋予糖基化化合物酸性特征。 Sialic acid is usually N- and O- linked oligosaccharides terminal residue, and due to its characteristics carries a negative charge, may confer acidic character glycosylated compound. 这些位点可以整合到本发明的化合物接头上,并优选在多肽化合物通过细胞重组生产时糖基化(例如,在哺乳动物细胞中,如CHO,BHK,COS)。 These sites can be incorporated into a joint compound of the present invention, preferably the polypeptide and the compound of group (e.g., in mammalian cells, such as CHO, BHK, COS) by a cell during recombinant production of sugar. 当然,这些位点也可以在本领域熟知的合成或半合成过程中糖基化。 Of course, these sites may be well known in the art of synthetic or semi-synthetic procedures glycosylation.

本发明的一些示例肽结构显示如下。 Some examples of the structure of the peptides of the present invention are shown below. 其中应用的是氨基酸单个字母缩写形式,并且为清晰起见,接头用横线分开表示。 Wherein the application is a single letter abbreviation of amino acids, and for clarity are shown separately with a line joint. 其他缩写:BrAc为溴乙酰(BrCH2C(O)),PEG为聚乙二醇。 Other abbreviations: BrAc is a bromoacetyl (BrCH2C (O)), PEG is polyethylene glycol.

IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (序列10) IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (10 sequences) (序列11)IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA(线性)(序列12)IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (序列13)IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA (序列14)IEGPTLRQWLAARA-GGGK(BrAc)GGGG-IEGPTLRQWLAARA(序列15)IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (序列16)IEGPTLRQWLAARA-GGGK(PEG)GGGG-IEGPTLRQWLAARA(序列17)IEGPTLRQWLAARA-GGGC(PEG)GGGG-IEGPTLRQWLAARA(序列18)IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (序列19)IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA|IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (序列20)IEGPTLROWLAARA-GGGGGGGG-IEGPTLRQWLAARA (序列21)上述每一种化合物中,N末端蛋氨酸(或M残基,用单个字母表示)也在考虑之列。 (SEQ ID 11) IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear) (SEQ ID 12) IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (sequence 13) IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA (sequence 14) IEGPTLRQWLAARA-GGGK (BrAc) GGGG-IEGPTLRQWLAARA (sequence 15) IEGPTLRQWLAARA-GGGCGGGG -IEGPTLRQWLAARA (sequence 16) IEGPTLRQWLAARA-GGGK (PEG) GGGG-IEGPTLRQWLAARA (sequence 17) IEGPTLRQWLAARA-GGGC (PEG) GGGG-IEGPTLRQWLAARA (sequence 18) IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (sequence 19) IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA | IEGPTLRQWLAARA-GGGCGGGG -IEGPTLRQWLAARA (sequence 20) IEGPTLROWLAARA-GGGGGGGG-IEGPTLRQWLAARA (sequence 21) in each of the above compound, N-terminal Met (or M residue, represented by a single letter) were also considered. 上述化合物的聚合物(例如,串联或非串联,共价键连接或非共价键连接)也在考虑之列。 The above-described polymer compound (e.g., in series or in series, or non-covalent bond covalent bond) are also contemplated.

在本发明的第二个实施方案中,上述化合物还可进一步与一个或多个Fc基团直接或通过接头基团融合。 In a second embodiment of the present invention, the above compounds may further fusion Fc group either directly or via a linker group to one or more. 通常,这个第二组化合物的结构式为:(Fc)m-(L2)q-TMP1-(L1)n-TMP2-(L3)r-(Fc)p其中和TMP1,TMP2和n分别与上述相同;L1,L2和L3是分别独立选自上述接头基团的接头基团;Fc是免疫球蛋白的Fc段;m,p,q和r是分别独立选自0和1的数字,其中m或p至少有一个是1,而且,如果m为0则q为0,如果p为0则r为0;及其生理学可以接受的盐类。 Typically, the formula of this second group of compounds is: (Fc) m- (L2) q-TMP1- (L1) n-TMP2- (L3) r- (Fc) p and wherein TMP1, TMP2 and n are each as defined above ; L1, L2 and L3 are each independently a linker group selected from the linker groups described above; Fc is the Fc portion of an immunoglobulin; m, p, q and r are each independently selected from the numbers 1 and 0, or wherein m p is 1, at least one, and, if m is 0 then q is 0, p is 0 if r is 0; and physiologically acceptable salts thereof.

据此,这个第二组的化合物具有上述第一组化合物定义的结构,但是,该化合物进一步与至少一个Fc基团直接或通过一个或多个接头融合。 Accordingly, this second group of compounds having the structure of a first group of compounds defined above, however, the compound further at least one Fc group either directly or with fusion via one or more linkers.

上述化合物的Fc序列可选自人免疫球蛋白IgG-1的重链,见Ellison,JW等,核酸研究,10:4071-4079(1982),或本领域熟知的任意Fc序列(例如,其他IgG类型,包括但不限于IgG-2,IgG-3和IgG-4,或其他免疫球蛋白)。 Fc sequence of the above compounds can be selected from the human immunoglobulin IgG-1 heavy chain, see Ellison, JW et al., Nucleic Acids Res., 10: 4071-4079 (1982), or any sequence Fc (e.g., IgG others known in the art types, including but not limited to IgG-2, IgG-3 and IgG-4, or other immunoglobulins).

众所周知,抗体的Fc段由单体多肽片段组成,这些单体多肽片段可以通过二硫键或非共价键连接构成二聚体或多聚体形式。 Is well known, the Fc polypeptide from a monomer segment antibody fragments, polypeptide fragments of these monomers may be connected by disulfide bonds or non-covalent dimeric or multimeric form. 在天然Fc分子的单体亚单位间形成的分子内二硫键数量从1-4不等,有赖于参与抗体的类型(如IgG,IgA,IgE)或亚型(如IgG-1,IgG-2,IgG-3,IgA-1,IgA-2)。 Number of intramolecular disulfide bonds formed between monomeric subunits of native Fc molecules ranges from 1-4, depend on the type (e.g., IgG, IgA, IgE) or participation of the antibody subtypes (e.g., IgG-1, IgG- 2, IgG-3, IgA-1, IgA-2). 这里所用的术语“Fc”可以代表Fc分子的单体,二聚体和多聚体形式。 As used herein, the term "Fc" Fc molecule may represent monomeric, dimeric and multimeric form. 应当注意,当适宜的半胱氨酸残基存在时,Fc单体会自动形成二聚体,除非存在阻止二硫键形成从而阻止二聚体产生的特定条件。 It should be noted that, when the presence of a suitable cysteine ​​residue, the Fc monomer may form a dimer automatically, unless there is a specific condition thereby preventing disulfide bond formation of dimers blocked. 即使在Fc二聚体中可以正常形成二硫键的半胱氨酸被去除或被其他残基取代,单体链通常也可通过非共价作用二聚体化。 Disulfide bond can be formed even if the cysteines are removed or substituted with other residues, the monomeric chain also generally dimerize through non-covalent interactions in normal Fc dimer. 这里的术语“Fc”用来表示下述任一形式:天然单体,天然二聚体(二硫键连接),修饰二聚体(二硫键和/或非共价连接),和修饰单体(即衍生物)。 Here, the term "Fc" is used to indicate any of the following forms: the native monomer, the native dimer (disulfide linked), modified dimers (disulfide and / or non-covalently linked), and modified single (i.e. derivatives thereof).

Fc部分的变异体、类似物或衍生物可以通过,例如进行残基或序列的多种取代来构建。 Variant Fc portion, analogue or derivative can be, for example, a variety of substituted residues or sequences constructed.

变异体(或类似物)多肽包括插入变异,其中一个或多个氨基酸残基增补到Fc氨基酸序列中。 Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues supplement to the Fc amino acid sequence. 插入位置可以是蛋白质的任一末端或双末端,也可以是Fc氨基酸序列的内部区域。 Insertion position can be at either end or both ends of the protein, or may be an internal region of the Fc amino acid sequence. 在任一末端或双末端添加残基得到的插入变异体可以包括,如融合蛋白和包括氨基酸标志或标记的蛋白质。 At either end or both ends added resulting residue insertion variants may include, as a fusion protein and comprises an amino acid or labeled protein markers. 例如,Fc分子可以可选地包括一个N末端蛋氨酸,特别是当分子在细菌体如大肠杆菌中重组表达时。 For example, the Fc molecule may optionally include a N-terminal methionine, especially when the molecule in bacteria such as E. coli bodies when recombinantly expressed.

在Fc缺失变异体中,Fc多肽中的一个或多个氨基酸残基被去除。 In Fc deletion variants, Fc polypeptide with one or more amino acid residues are removed. 缺失可以在Fc多肽的一端或两端发生,也可在Fc氨基酸序列内除去一个或多个残基。 Deletion may occur at one or both ends of the Fc polypeptide, may also be the removal of one or more residues within the Fc amino acid sequence. 因此,缺失变异体包括Fc多肽序列的所有片段。 Thus, deletion variant Fc polypeptide comprises a sequence of all fragments.

在Fc取代变异体中,Fc多肽的一个或多个氨基酸残基被去除并被其他残基所取代。 In Fc substitution variants, one or more amino acid residues in an Fc polypeptide are removed and replaced by other residues. 一方面,自然发生的取代是保守的,但本发明也包括非保守取代。 In one aspect, the substitution is conservative naturally occurring, but the present invention also encompasses non-conservative substitutions.

例如,为了阻止在Fc序列中形成一些或全部二硫键交联,可以去除半胱氨酸残基或用其他氨基酸取代。 For example, in order to prevent formation of some or all disulfide crosslinks of the Fc sequence, a cysteine ​​residue may be removed or substituted with other amino acids. 特别是,在序列5中,第7和第10位氨基酸是半胱氨酸残基。 In particular, the sequence in the 5, 7 and 10 amino acids is a cysteine ​​residue. 可以将这些半胱氨酸残基各个去除,也可以用其他氨基酸,如丙氨酸或丝氨酸取代这些半胱氨酸残基中的一个或多个。 These cysteine ​​residues can be removed each, other amino acids may also be used, such as alanine or serine substitution of one or more of these cysteine ​​residues. 如另一实例所示,修饰还可以通过引入氨基酸取代(1)去除Fc受体结合位点;(2)去除补体(Clq)结合位点;和/或(3)去除抗体依赖型细胞介导的细胞毒作用(ADCC)位点。 As another example, the modified amino acid may also be substituted by the introduction of (1) removing the Fc receptor binding site; (2) removing the complement (CIq) binding site; and / or (3) removing the antibody-dependent cell-mediated cytotoxicity (ADCC) site. 这些位点在本领域是众所周知的,而且Fc范围内任何熟知的取代都可采用。 These sites are well known in the art, and any known substitutions can be employed within the scope of Fc. 例如,见分子免疫学,29卷,第5期,633-639(1992),就是IgG1上的ADCC位点。 For example, see Molecular Immunology, Vol. 29, No. 5, 633-639 (1992), it is the site on ADCC IgG1.

同样,一个或多个酪氨酸残基也可由苯丙氨酸残基取代。 Likewise, one or more tyrosine residues can also be substituted with a phenylalanine residue. 此外,其他氨基酸插入、缺失(例如从1-25氨基酸)和/或取代变异体也在考虑之列,包括在本发明范围之内。 In addition, other amino acid insertions, deletions (e.g., from 1-25 amino acids) and / or a substituted variant thereof column also contemplated, within the scope of the present invention. 通常优选保守的氨基酸取代。 Conservative amino acid substitutions are generally preferred. 另外,改变可以是氨基酸形式的改变,如模拟肽或D-氨基酸。 Further, the change may be in the form of amino acid changes, such as peptidomimetics or D- amino acids.

TMP化合物的Fc序列也可以衍生,即修饰不仅限于氨基酸残基的插入、缺失或取代。 Fc sequence TMP compound may also be derivatized, i.e., is not limited to modification of amino acid residues inserted, deleted or substituted. 优选的修饰是天然存在的共价连接,包括如与聚合物、脂质、其他有机和无机部分的化学连接。 Preferred modifications are naturally occurring covalent linkage, such as a connection comprising polymers, lipids, other organic and inorganic chemical moieties. 本发明的衍生物可制备成延长循环半衰期,或设计成改善多肽对期望细胞、组织或器官的靶向定位能力。 Derivatives of the invention may be prepared to prolong circulating half-life, or designed to improve the desired polypeptide on the cell, tissue or organ targeting ability of positioning.

还可应用完整Fc分子的救援受体结合域作为本发明化合物的Fc部分,如在题为“具有延长半衰期的改变多肽”的专利WO96/32478中所述。 May also be applied intact Fc molecule as the Fc domain relief receptor binding moiety of the compound of the present invention, as in the patent entitled "polypeptide having a half-life extending change" in the WO96 / 32478. 在这里命名为Fc分子类型的其他成员是在题为“具有延长半衰期的免疫球蛋白样域”的专利WO97/34631中所述的分子。 Herein designated as type Fc molecule other members are entitled "having a half-life extending immunoglobulin-like domain" of a molecule of the patent WO97 / 34631. 本段中引用的两个公开发表的PCT申请,在此引入,作为参考。 PCT application cited in this paragraph are two published, incorporated herein by reference.

Fc融合可以发生在TMP1或TMP2的N末端或C末端,或者发生在TMPs的N和C两端。 Fc fusion TMP1 or TMP2 may occur at the N-terminus or C-terminus, or at both ends of C and N occurs in the TMPs. 令人惊异的发现是,Fc部分连接于TMP基团N末端的肽较其他可能性具有更高的生物活性,因此优选在TMP1N末端拥有Fc功能域的融合体(即在通式中,r和p同时为0,而m和q同时为1)。 The finding is surprising, Fc peptide moiety is attached to the N-terminus of the TMP group other than the possibility of having a higher biological activity, it is preferable to have the Fc fusion domain in TMP1N end (i.e., in the general formula, r and p simultaneously 0, and m and q are 1). 当Fc链与TMP或接头的N末端融合时,这种融合通常发生在Fc链的C末端,反之亦然。 When the Fc chain N-terminal fusion TMP or linker, such fusion occurs at the C-terminus is usually Fc chain, and vice versa.

同样优选的化合物是上述通式公布的化合物二聚体(例如,串联和非串联)。 Also preferred compounds are compounds of the above general formula published dimers (e.g., tandem and non-tandem). 在这些情况下,每个Fc链都与一个串联TMP肽二聚体相连。 In these cases, each Fc chain is connected in series with a peptide dimer linked TMP. 这样一种化合物的图例在图6C中显示。 Legend of such a compound is shown in Figure 6C. 这种类型化合物的优选实例基于图6C,其中,Fc是序列5化合物的二聚体,L2是(甘氨酸)5,TMP1和TMP2是序列1的化合物,L1是(甘氨酸)8。 Preferred examples of compounds of this type based to Figure 6C, where, Fc is a dimer compound 5 sequence, L2 is (Gly) 5, TMP1 and TMP2 are the compounds of the sequence 1, L1 is (Gly) 8. 这种化合物在这里也称之为“Fc-TMP1-L-TMP2”。 Such compounds are also referred to herein "Fc-TMP1-L-TMP2". 它也可以序列34的二聚体(通过Fc部分)形式表现。 It may also be 34 sequences dimer performance (via the Fc part) form. 在图6C中所示,Fc基团通过接头与TMP2基团的C末端连接的化合物类似物,也在考虑之列,并在这里称之为“TMP1-L-TMP2-Fc”。 Shown in FIG. 6C, the Fc group like the compound C-terminal linker TMP2 groups attached, it was also considered, and referred to herein as "TMP1-L-TMP2-Fc".

第二组的一些特例化合物在下面给出:Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (序列22)Fe-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA-Fc (序列23)IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA-Fc (序列24)Fc-GG-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (序列25)Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (序列26) Specific examples of some compounds in the second group given below: Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (sequence 22) Fe-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA-Fc (sequence 23) IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA-Fc (sequence 24) Fc-GG -IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (sequence 25) Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (26 sequences) (序列27)Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA(线性)(序列28)Fc-IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (序列29)Fc-IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA (序列30)Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (序列31)Fc-IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (序列32)Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA|Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (序列33)Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA(序列34)上述每一种化合物中,添加N末端蛋氨酸(或M残基,用单个字母表示)也在考虑之列。 (SEQ ID 27) Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear) (SEQ ID 28) Fc-IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (sequence 29) Fc-IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA (sequence 30) Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (Sequence 31 ) Fc-IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (sequence 32) Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA | Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (sequence 33) Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (sequence 34) in each of the above compound was added N-terminal methionine (or M residue, represented by a single letter) were also considered. 在这些情况下,蛋氨酸残基可以与Fc基团的N末端连接,其中,Fc基团与TMP的N末端相连。 In these cases, a methionine residue may be linked to the N terminus of the Fc group, wherein, Fc group is attached to the N-terminus of the TMP. 在另一些情况下,Fc基团与TMP的C末端相连,蛋氨酸残基可以与TMP基团的N末端连接。 In other cases, the Fc group attached to the C-terminus of the TMP, a methionine residue may be linked to the N-terminus of the TMP group.

在每种上述情况下,Fc优选人免疫球蛋白IgG1重链的Fc段,或其生物活性片段、衍生物或二聚体,见Ellison,JW等,核酸研究,10:4071-4079(1982)。 In each case above, preferably Fc segment of human immunoglobulin Fc IgG1 heavy chain or a biologically active fragment, derivative or dimer, see Ellison, JW et al., Nucleic Acids Res., 10: 4071-4079 (1982) . 序列5所示的Fc序列是上述化合物最优选的Fc段。 Fc sequence shown in Sequence 5 is the most preferred compound of the above-described Fc region. 同样优选的化合物是,Fc为序列5的二聚体形式,并且每一Fc链与一个TMP串联二聚体相连的化合物。 Also preferred compounds are, dimeric form Fc sequence 5 and each Fc chain is attached to a compound of a TMP tandem dimer.

此外,尽管第二个实施方案的很多优选化合物包括一个或多个拥有两个相连的TMP部分的串联二聚体,本发明的其他化合物还包括串联TMPs多聚体,即如下示例结构的化合物:Fc-TMP1-L-TMP2-L-TMP3;Fc-TMP1-L-TMP2-L-TMP3-L-TMP4;Fc-TMP1-L-TMP2-L-TMP3-L-TMP4-L-TMP5;TMP1-L-TMP2-L-TMP3-L-Fc;TMP1-L-TMP2-L-TMP3-L-TMP4-L-Fc;TMP1-L-TMP2-L-TMP3-L-TMP4-L-TMP5-L-Fc;其中,TMP1,TMP2,TMP3,TMP4和TMP5的结构可以相同,也可以不同,并且Fc,TMP和L的定义如上所述,接头是可选的。 In addition, although many preferred compounds of the second embodiment includes one or more of TMP tandem dimer has two connected portions, other compounds of the present invention further includes a series multimeric TMPs, i.e., compounds of the following exemplary structures: Fc-TMP1-L-TMP2-L-TMP3; Fc-TMP1-L-TMP2-L-TMP3-L-TMP4; Fc-TMP1-L-TMP2-L-TMP3-L-TMP4-L-TMP5; TMP1- L-TMP2-L-TMP3-L-Fc; TMP1-L-TMP2-L-TMP3-L-TMP4-L-Fc; TMP1-L-TMP2-L-TMP3-L-TMP4-L-TMP5-L- FC; wherein, TMP1, TMP2, TMP3, TMP4, and TMP5 structure may be the same or different, and Fc, TMP and L are as defined above, the linker is optional. 在上述每种情况下,Fc基团可以是单体,或二聚体,并且当Fc是二聚体时,一个或多个TMP多聚体可与每条Fc链相连。 In each of these cases, Fc group can be monomeric, or dimeric, and when the Fc is dimeric, one or more TMP multimer can be connected to each Fc chains. 其他实例也在考虑之列,其中TMP二聚体或多聚体与一条或两条Fc链的N与C末端同时相连,包括TMP二聚体或多聚体与两条Fc链的所有四个末端相连的情况。 Other examples were also considered, where N and C-terminus of the TMP dimer or multimer of one or both Fc chains connected while TMP dimer or multimer comprising two Fc chains with all four end is connected to the case.

优选,本发明第二个实施方案的优选化合物总共有大约200-400个氨基酸(即,他们是多肽)。 Preferably, a second embodiment of the preferred compounds of the present invention, a total of approximately 200-400 amino acids (i.e., they are polypeptide).

制备方法本发明的化合物可通过多种方式制备。 The method of preparation of compounds of the present invention may be prepared by a variety of ways. 因为很多化合物是肽,或包括肽,因此肽合成方法在这里尤其重要。 Because many compounds are peptides, including peptides or, peptide synthesis methods therefore particularly important here. 例如,可能用到固相合成技术。 For example, solid phase synthesis techniques may be used. 适宜的技术在本领域是熟知的,并包括以下文献描述的方法,包括Merrifield,化学肽,335-61页(Katsoyannis和Panayotis编辑,1973);Merrifield,美国化学学会杂志,85:2149(1963);Davis等,国际生物化学杂志,10:394-414(1985);Stewart和Young,固相肽合成(1969);美国专利3941763;Finn等,蛋白质,第三版,第二卷,105-253页(1976);以及Erickson等,蛋白质,第三版,第二卷,257-527页(1976)。 Suitable techniques are well known in the art, and include the following methods described in the literature, including Merrifield, chemical peptide, pages 335-61 (Katsoyannis and Panayotis editor, 1973); Merrifield, Journal of the American Chemical Society, 85: 2149 (1963) ; Davis et al., international Journal of Biochemistry, 10: 394-414 (1985); Stewart and Young, solid phase peptide synthesis (1969); U.S. Patent No. 3941763; Finn et al., proteins, Third Edition, volume II, 105-253 (1976); and Erickson et al, proteins, Third Edition, volume II, pages 257-527 (1976). 固相合成法是制备各种肽的优选技术,因为这种方法是制备小型肽最经济有效的方法。 Solid phase synthesis is the preferred technique for preparing various peptides, because this method is the preparation of small peptides of the most cost-effective way.

所述肽还可通过重组DNA技术在转化宿主细胞中制备。 The peptide may also be prepared in a host cell transformed by recombinant DNA techniques. 为达成此目的,需要制备编码该蛋白的重组DNA分子。 To achieve this purpose, the preparation of a recombinant DNA molecule encoding the protein. 制备这种DNA和/或RNA的方法在本领域是熟知的。 Preparing such DNA and / or RNA, it is well known in the art. 例如,编码所述肽的序列可应用合适的限制性酶从DNA中切除。 For example, a sequence encoding the peptide can be applied to a suitable restriction enzyme excised from the DNA. 相关序列可应用包括有用限制性位点的聚合酶链反应(PCR)扩增创建,以备后续克隆。 Related applications include sequences may be useful restriction sites polymerase chain reaction (PCR) amplification to create, for subsequent cloning. 此外,DNA/RNA分子还可通过化学合成技术合成,例如亚磷酰胺法。 In addition, DNA / RNA molecule may by chemical synthesis techniques, e.g. phosphoramidite method. 而且,还可将这些技术组合使用。 Furthermore, these techniques may also be used in combination.

本发明还包括一种在适宜宿主中编码肽的载体。 The present invention also includes a vector in a suitable host the peptide coded. 载体包括可操作性连接于适宜表达调控序列的编码肽DNA分子。 Vector comprising a operably linked to an appropriate expression control sequence encoding a peptide DNA molecule. 无论在肽编码DNA分子插入载体之前或之后,影响这一操作性连接的方法众所周知。 Whether a DNA molecule encoding a peptide before or after inserted into the vector, operably linked to the influence of this method are well known. 表达调控序列包括启动子,激活物,增强子,操纵子,核糖体结合位点,起始信号,终止信号,戴帽信号,聚腺苷化信号,以及其他在转录或翻译过程中起调控作用的信号。 Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals wearing, polyadenylation signals, and others from the process of transcription or translation regulation signal of.

用得到的包含肽编码DNA分子的载体转化适宜宿主。 With a vector comprising a DNA molecule encoding a peptide obtained by transforming a suitable host. 该转化过程可用本领域熟知的方法进行。 This conversion process is well known in the art can be used.

在大量可以通过商业途径得到和众所周知的宿主细胞中,任何一种都可用于本发明实践。 In a large number of host cells are well known and can be obtained commercially in any one it can be used in the practice of the present invention. 选择一种特定宿主有赖于本领域业已认定的多种因素。 Select a particular host depends on a variety of factors have been identified in the art. 这些因素包括,例如,与所选表达载体的兼容性,DNA分子编码的肽对宿主细胞的毒性,转化率,回收肽的容易程度,表达特性,生物安全性和花费。 These factors include, for example, compatibility with the chosen expression vector, DNA molecule, encoding a peptide toxic to the host cell, conversion rate, ease of recovery of the peptides, expression characteristics, biosafety and costs. 必须平衡这些因素,而且应该理解,对于一个特定DNA序列的表达,并非所有宿主都有相等的效力。 These factors must be balanced, and it should be understood that, for the expression of a particular DNA sequence, not all hosts have equal efficacy.

在这些一般原则的指导下,有用的微生物宿主包括细菌(如大肠杆菌),酵母菌(如酵母类,巴斯德毕赤酵母)和其他真菌,昆虫,植物,培养的哺乳动物(包括人类)细胞,或其他本领域熟知的宿主。 Guided by these general principles, useful microbial hosts include bacteria (such as E. coli), yeast (e.g., yeasts, Pichia pastoris) and other fungi, insect, plant, cultured mammalian (including human) cells, or other hosts known in the art.

然后,转化宿主在常规发酵条件下培养,使所需肽表达。 Then, the transformed host cultured under conventional fermentation conditions so that the desired peptide expression. 这些发酵条件在本领域是众所周知的。 The fermentation conditions are well known in the art.

最后,从表达所述肽的发酵培养基或宿主细胞中纯化所述肽。 Finally, the peptide is purified from the fermentation medium or host cell expressing said peptide. 这些纯化方法在本领域也是众所周知的。 These purification methods are also well known in the art.

含有衍生肽或非肽基团的化合物可通过众所周知的有机化学技术合成。 Derived peptide or non-peptide compounds containing groups may be synthesized by well-known organic chemistry techniques.

化合物的应用本发明化合物具有与c-Mpl受体结合并激活它的功能,和/或具有刺激(无论体内和体外)血小板产生(血小板生成活性)和血小板前体产生(巨核细胞生成活性)的能力。 Compound applications of the present invention is a compound having binding and activating its function and c-Mpl receptor, and / or stimulate (whether in vivo and in vitro) platelets (thrombopoietic activity) form generation and platelets before (megakaryocytopoiesis activity) ability. 可应用标准方法,如在题为“刺激巨核细胞生长分化的组合物和方法”的WO95/26746中描述的方法,来评价这些化合物的功能。 WO95 may be applied by standard methods, such entitled "compositions and methods for stimulating megakaryocyte growth differentiation" method described in 26746 /, to evaluate the functionality of these compounds. 体内试验将在实施例部分给予进一步描述。 Test administered in vivo is further described in the Examples section.

应用本发明组合物和方法治疗的病况通常是已经存在的巨核细胞/血小板减少,或在未来预期或期望的巨核细胞/血小板减少(如由于计划进行的手术或捐献血小板)。 Application of the composition and condition treated in the present methods is generally already existing megakaryocyte / platelet reduction, or reducing (e.g., due to surgery or platelet donation planned) in the future expected or desired megakaryocytes / platelets. 这些病况可能是由于体内活性Mpl(暂时性或永久性)缺乏导致。 These conditions may be due to in vivo activity of Mpl (temporary or permanent) deficiency leads. 描述血小板减少的术语为血小板缺乏症,本发明的组合物和方法通常可用来预防或治疗有需要的血小板缺乏症患者。 The term is described thrombocytopenia thrombocytopenia, the compositions and methods of this invention are generally useful in preventing or treating thrombocytopenia in patients in need.

世界卫生组织根据个体循环血小板的数量对血小板缺乏症进行了分类(Miller等,癌症,47:210-211(1981))。 World Health Organization classified according to the number of circulating platelets individual platelet deficiency (Miller et al., Cancer, 47: 210-211 (1981)). 例如,没有血小板缺乏症体征的个体(0级)血小板计数通常至少为100 000/mm3。 For example, no signs of thrombocytopenia individuals (0) platelet count is generally at least 100 000 / mm3. 轻度血小板缺乏症(1级)表示循环血小板水平为79 000-99 000/mm3。 Mild thrombocytopenia (Grade 1) indicates a level of circulating platelets 79 000-99 000 / mm3. 中度血小板缺乏症(2级)血小板水平为50 000-74 000/mm3,重度血小板缺乏症的特征是血小板计数为25 000-49 000/mm3。 Moderate thrombocytopenia (Grade 2) platelet levels of 50 000-74 000 / mm3, severe thrombocytopenia is characterized by platelet count 25 000-49 000 / mm3. 致死性或无力性血小板缺乏症的特征为循环血小板浓度低于25 000/mm3。 Platelet fatal weakness or deficiency of circulating platelets wherein concentration of less than 25 000 / mm3.

血小板缺乏症(血小板减少)可以由多种原因导致,包括化疗和其他应用多种药物的治疗,放疗,手术,意外失血,和其他具体疾病。 Platelet deficiency (thrombocytopenia) can lead to a variety of reasons, including chemotherapy and other applications of a variety of drugs, radiation therapy, surgery, accidental blood loss, and other specific diseases. 包括血小板缺乏症并可依据本发明进行治疗的具体疾病的示例为:再生障碍性贫血;特发性或免疫性血小板减少性紫癜(ITP);HIV相关性ITP和HIV相关性血栓形成性血小板减少性紫癜;导致血小板缺乏症的转移性肿瘤;系统性红斑狼疮;包括新生儿狼疮综合征的脾大;范可尼综合征;维生素B12缺乏症,叶酸缺乏症;May-Hegglin异常;Wiskott-Aldrich综合征;慢性肝病;与血小板缺乏症相关的骨髓增生异常综合征;阵发性睡眠性血红蛋白尿;C7E3Fab(Abciximab)治疗后的急性重症血小板缺乏症;同种异体免疫性血小板缺乏症,包括母体同种异体免疫性血小板缺乏症;与抗磷脂抗体和血栓形成相关的血小板缺乏症;自身免疫性血小板缺乏症;药物诱导的免疫性血小板缺乏症,包括卡铂诱导的血小板缺乏症,肝素诱导的血小板缺乏症;胎儿血小板缺乏症;妊娠性血小板缺乏 Including platelet deficiency Keyijuben invention and exemplary of the particular disease undergoing therapy are: aplastic anemia; idiopathic or immune thrombocytopenic purpura (ITP); HIV-associated ITP and HIV-related thrombotic thrombocytopenic purpura; metastatic tumor resulting in platelet deficiency; systemic lupus erythematosus; including neonatal lupus syndrome splenomegaly; Fanconi syndrome; vitamin B12 deficiency, folic acid deficiency; may-Hegglin anomaly; Wiskott-Aldrich syndrome; chronic liver disease; platelet deficiency associated with myelodysplastic syndrome; paroxysmal nocturnal hemoglobinuria; after C7E3Fab (Abciximab) therapy for severe acute thrombocytopenia; allogeneic immune thrombocytopenia, including maternal allogeneic immune thrombocytopenia; formed with antiphospholipid antibodies and thrombosis-related thrombocytopenia; autoimmune thrombocytopenia; drug-induced immune thrombocytopenia, including carboplatin-induced thrombocytopenia, heparin-induced platelet deficiency; fetal thrombocytopenia; gestational thrombocytopenia 症;Hughes综合征;狼疮样血小板缺乏症;意外和/或大量失血;骨髓增生障碍;患有恶性疾病的血小板缺乏症患者;血栓形成性血小板减少性紫癜,包括在肿瘤患者中表现为血栓形成性血小板减少性紫癜/溶血性尿毒症综合征的血栓形成性微血管病;自身免疫性溶血性贫血;隐性空肠憩室穿孔;纯红再障;自身免疫性血小板缺乏症;流行性肾病;利福平相关性急性肾衰竭;Paris-Trousseau血小板缺乏症;新生儿同种异体免疫性血小板缺乏症;阵发性睡眠性血红蛋白尿;胃癌的血液学改变;儿童期溶血性尿毒症综合征;病毒感染相关性血液学表现,包括甲型肝炎病毒和CMV相关性血小板缺乏症。 Disease; Hughes syndrome; lupoid thrombocytopenia; accidental and / or massive blood loss; myeloproliferative disorders; with platelet deficiency in patients with malignant disease; thrombotic thrombocytopenic purpura, including performance in cancer patients is formed thrombus thrombocytopenic purpura / hemolytic uremic syndrome, thrombotic microangiopathy; autoimmune hemolytic anemia; recessive jejunal diverticulum perforation; PRCA; autoimmune thrombocytopenia; epidemic nephropathy; Lifestyle flat-associated acute renal failure; Paris-Trousseau thrombocytopenia; allogeneic neonatal immune thrombocytopenia; paroxysmal nocturnal hemoglobinuria; hematological changes of gastric cancer; childhood hemolytic uremic syndrome; viral infection related hematologic manifestations, including hepatitis A virus and CMV-related platelet deficiency. 此外,艾滋病的一些治疗也可导致血小板缺乏症(如AZT)。 In addition, some AIDS treatment can also lead to platelet deficiency (such as AZT). 某些皮损愈合障碍也可受益于血小板数量的增加。 Some skin lesions heal disorders may also benefit from increased platelet counts.

对于预期的血小板减少,如由于将来的手术,可在血小板需要前几天或几小时给予本发明化合物。 Thrombocytopenia expectations, since such future surgery, may be days or hours before the administration of the compounds of the present invention need platelets. 对于紧急情况,如意外和大量失血,本发明化合物可与全血或纯化血小板一同给予。 For emergencies, such as accidental and massive blood loss, a compound of the present invention may be administered together with whole blood or purified platelets.

本发明化合物还可用于刺激巨核细胞以外的特定细胞类型,如果发现这些细胞表达Mpl受体的话。 Compounds of the invention may also be used to stimulate specific cell types other than megakaryocytes if such cells express Mpl receptor found words. 与这些表达Mpl受体细胞相关的病况,所述细胞对Mpl配体刺激有反应,也属于本发明范畴。 Associated with these conditions expression of Mpl receptor cells, the cells respond to Mpl ligand stimulation, also within the scope of the present invention.

本发明化合物还可用于以下任何情况,包括希望产生血小板或血小板前体细胞,或希望刺激c-Mpl受体。 Compounds of the invention may also be used in any of the following, including desirable to produce platelets or platelet precursor cells, or stimulate the desired c-Mpl receptor. 因此,例如,本发明化合物可用于哺乳动物需要血小板、巨核细胞等治疗的任何病况。 Thus, for example, the compounds of the present invention may be used in any condition in a mammal in need of platelets, megakaryocytes and other treatment. 这些病况在下述示例资料中有详细描述:WO95/26746;WO95/21919;WO95/18858和WO95/21920,在此引入。 In these conditions there are described in detail in the following exemplary materials: WO95 / 26746; WO95 / 21919; WO95 / 18858 and WO95 / 21920, herein incorporated.

本发明化合物对维持储藏期血小板和/或巨核细胞以及相关细胞的活性,也非常有益。 Compounds of the invention maintain the shelf-life of platelets and megakaryocytes or activity and / related cells, but also very useful. 因此,在含有这些细胞的组合物中包括一种或多种这种有效剂量的化合物,是很有用的。 Thus, in compositions containing these cells comprise one or more compounds of such an effective dose, it is useful.

“哺乳动物”的意思是任何一种哺乳动物,包括人类,居家豢养的动物包括狗和猫;野外和/或动物园豢养的动物包括猴子;实验室的实验动物包括小鼠,大鼠和荷兰猪;农场养殖动物如马,牛,绵羊,山羊和猪等。 "Mammal" means any mammal, including humans, home reared animals, including dogs and cats; wild and / or feeding zoo animals including monkeys; laboratory animals including mice, rats and guinea pigs ; farmed animals such as horses, cattle, sheep, goats and pigs. 优选哺乳动物是人类。 Preferably the mammal is a human.

药用组合物本发明还提供应用本发明化合物构成药用组合物的方法。 Pharmaceutical compositions of the present invention also provides a method for forming a pharmaceutical composition of the compound of the present invention. 这些药用组合物可通过注射给药,或口服给药,鼻腔给药,经皮给药,或其他方式给药,包括,例如静脉给药,皮内给药,肌内给药,乳腺内给药,腹膜内给药,鞘内给药,眼内给药,球后给药,肺内给药(如雾化药物),或皮下注射给药(包括皮下埋植长期释放给药);通过舌下,肛门,阴道,或外科手术埋植,如在脾囊、脑、或角膜内植入给药。 Such as intravenous, intradermal, intramuscular, intramammary These pharmaceutical compositions may be administered by injection, or oral administration, nasal administration, transdermal administration, or administration by other means, including , intraperitoneal, intrathecal, intraocular administration, administration, retrobulbar intrapulmonary (e.g., aerosolized medicament), or subcutaneous injection (including long-term release implants administration); by sublingual, anal, vaginal, or surgical implants, such as in the spleen capsule, brain, or administered corneal implant. 治疗包括单剂给药或在一段时期内重复给药。 Treatment comprises administering a single or repeated administration over a period of time. 通常,本发明需要理解的是药用组合物包括有效剂量的一种本发明化合物与可药用的稀释剂,防腐剂,增溶剂,乳化剂,佐剂和/或载体。 Typically, the present invention is to be understood that a pharmaceutical composition comprising a compound of the invention with a pharmaceutically acceptable diluent present effective dose of one kind, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. 这些组合物包括多种缓冲液(如Tris-HCL,乙酸盐,磷酸盐),pH值和离子强度的稀释剂;添加剂如去污剂和增溶剂(如Tween 80,聚山梨醇酯80),抗氧化剂(如抗坏血酸,偏二亚硫酸钠),防腐剂(如硫柳汞,苯乙醇)和膨胀剂(如乳糖,甘露醇);将所述物质整合到聚合物化合物的特定制剂中,如聚乳酸,聚乙醇酸等,或整合到脂质体中。 These compositions include various buffers (e.g., Tris-HCL, acetate, phosphate), pH and ionic strength of the diluent; and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80) antioxidants (such as ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol) and bulking agents (e.g. lactose, mannitol); integrating said substance to a particular polymer compound in the formulation, such as polylactic acid, polyglycolic acid, etc., or incorporated into liposomes. 还可应用透明质酸,此点对于提高循环维持时间可能有效。 Can also be applied hyaluronic acid, this point may be effective for improving the cycle duration. 药用组合物还可可选地包括其他药物学可以接受的用作药物媒介物、赋形剂、或媒体的流体,半固体,或固体稀释剂,包括但不限于聚乙烯脱水山梨糖醇单月桂酸酯,硬脂酸镁,甲基-和丙基羟苯甲酸酯,淀粉,蔗糖,葡萄糖,阿拉伯胶,磷酸钙,矿物油,可可粉白脱,和可可油。 The pharmaceutical composition may further optionally include other pharmaceutically acceptable as pharmaceutical vehicles, excipients, or media fluid, semisolid, or solid diluents, including, but not limited to, polyethylene dehydrated sorbitan monolaurate esters, magnesium stearate, methyl - and propyl parabens, starch, sucrose, dextrose, gum acacia, calcium phosphate, mineral oil, cocoa Koshiro off, and cocoa butter. 这些组合物可以影响本发明蛋白质和衍生物的物理状态,稳定性,体内释放率和体内清除率。 These compositions may influence the protein derivatives of the invention and the physical state, stability, rate of in vivo release, and rate of in vivo clearance. 见,如雷明顿药理学,第18版(1990,Mack出版有限公司,Easton,巴拿马18042),1435-1712页,在此引入,作为参考。 See, eg Remington Pharmacology, 18th Edition (1990, Mack Publishing Co., Easton, Panama 18042), pages 1435-1712, incorporated herein by reference. 制备的组合物可以是液态形式,也可以是干粉形式,如冻干形式。 The composition may be prepared in liquid form, it may be in the form of a dry powder, such as lyophilized form. 植入式持续释放剂型也在考虑之列,如经皮剂型。 Implantable sustained release formulations are also taken into account, such as a transdermal dosage form.

这里考虑的应用剂型是口服固体剂量形式,在雷明顿药理学,第18版(Mack出版有限公司,Easton,巴拿马18042)第89章中有描述,该书在此引入,作为参考。 Application forms considered here is the solid oral dosage form, in Remington Pharmacology, 18th Edition (Mack Publishing Co., Easton, Panama 18042) are described in Chapter 89, the book is hereby incorporated by reference. 固体剂型包括片剂,胶囊,丸剂,锭剂或糖锭剂,扁胶囊剂或小糖丸。 Solid dosage forms include tablets, capsules, pills, troches or lozenges, capsules, flat or small pellets. 同样,脂质体或蛋白质样胶囊也可用于构建本发明组合物(例如,在美国专利4925673中报道的蛋白质样小丸)。 Similarly, liposomes or protein may also be used to construct the capsule-like compositions (e.g., U.S. Patent No. 4,925,673 reported in the pellet-like protein) of the present invention. 脂质体胶囊也可应用,并且脂质体可通过多种聚合物衍生获得(如美国专利5 013 556)。 Liposomal encapsulation may be applied, and liposomes may be obtained by a variety of polymer derivatives (e.g., U.S. Patent No. 5,013,556). 药物可能的固体剂型在1979年出版的由GSBanker和CTRhodes编辑的现代药理学第10章中由Marshall,K.给出描述,在此引入,作为参考。 Drug possible solid dosage forms in Chapter 10 modern pharmacology, published in 1979 by the GSBanker and CTRhodes edited by Marshall, K. Description is given, which is incorporated herein by reference. 通常,制剂包括本发明化合物以及惰性成分,这些成分可以抵御胃酸环境,并在小肠内释放生物活性物质。 Typically, formulations comprising a compound of the invention and inert ingredients that can withstand gastric acid environment, and release of the biologically active substance in the small intestine.

同样给予考虑的是上述本发明化合物的口服剂型。 Also contemplated are oral dosage forms given above compounds of the present invention. 如有需要,化合物可进行化学修饰使口服有效。 If necessary, the compounds may be chemically modified so that oral valid. 通常,考虑的化学修饰是在化合物分子上连接至少一个部分,其中所述部分允许(a)抑制蛋白水解;以及(b)从胃和肠壁摄取进入血流。 Generally, the chemical modification contemplated is linked to at least a portion in the compound molecule, wherein said moiety permits (a) inhibition of proteolysis; and (b) uptake from the stomach and the intestinal wall into the bloodstream. 同样需要的是增加化合物的整体稳定性并延长体内循环时间。 Also needed is to increase the overall stability of the compound and to extend circulation time in vivo. 这些部分的示例包括:聚乙二醇,乙二醇和丙二醇共聚物,羧甲基纤维素,葡萄糖,聚乙烯醇,聚乙烯吡咯烷和聚脯氨酸(Abuchowski和Davis,可溶性聚合物酶加合物,药物酶,Hocenberg和Roberts编辑,Wiley-Interscience,纽约,纽约州,(1981),367-383页;Newmark等,实用生物化学杂志,4:185-189(1982))。 Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, glucose, polyvinyl alcohol, polyvinyl pyrrolidine and polyproline (Abuchowski and Davis, Soluble Polymer Enzyme additive , drugs enzyme, Hocenberg and Roberts edited, Wiley-Interscience, New York, New York, (1981), pages 367-383; Newmark et al., Journal of Biological Chemistry practical, 4: 185-189 (1982)). 其他可用的聚合物是聚-1,3-diaxolane和聚-1,3,6-tioxocane。 Other useful polymers are polypropylene and polyethylene -1,3-diaxolane -1,3,6-tioxocane. 如上所示,优选的药用部分是聚乙二醇。 As indicated above, preferred pharmaceutical moiety is a polyethylene glycol.

作为口服剂型,还可应用修饰脂肪族氨基酸的盐,如N-(8-[2-羟苯]氨)辛酸钠(SNAC),作为增加本发明治疗性化合物吸收的载体。 As an oral dosage form, it may also be applied a modified aliphatic amino acid salts, such as N- (8- [2- hydroxyphenyl] amino) caprylate (of SNAC), as a carrier to increase the therapeutic compounds of the invention is absorbed. 应用SNAC肝素剂型的临床有效性业已由Emisphere Technologies进行的II期临床试验显示。 Phase II clinical trials clinical efficacy of heparin dosage forms application SNAC has been conducted by Emisphere Technologies display. 见美国专利5 792 451,“口服药物的给药组合物和方法”。 See U.S. Patent No. 5,792,451, "Oral administration of the pharmaceutical compositions and methods."

治疗药物剂型还可包括由颗粒大小约为1mm的颗粒或小糖丸形式构成的细小多颗粒。 Treatment may further comprise pharmaceutical dosage form of fine particles composed of a multi-particle size of about 1mm in the form of particles or pellets. 以胶囊形式给药的剂型还可做成粉剂,轻度压缩的栓剂甚或片剂。 The dosage administered may be made in the form of a capsule, powders, suppositories or even slightly compressed tablets. 治疗药物可通过压缩技术制备。 Therapeutic agent can be prepared by compression techniques.

色素和调味料也可都包含在内。 Colorings and flavorings are also included. 例如,蛋白质(或衍生物)可以配制在内(如通过脂质体或微球胶囊),然后再添加可食性产品,如包括色素和调味料的冷冻液体。 For example, the protein (or derivative) may be formulated into account (such as by liposome or microsphere capsules), and then add edible products, such as frozen liquid comprising a dye and flavoring.

还可应用惰性物质稀释或增加治疗药物的体积。 May dilute or increase the volume of the therapeutic agent with inert material. 这些稀释剂可包括碳水化合物,特别是甘露醇,α-乳糖,无水乳糖,纤维素,蔗糖,修饰的葡萄糖和淀粉。 These diluents could include carbohydrates, especially mannitol, alpha] lactose, anhydrous lactose, cellulose, sucrose, modified starches and glucose. 某些无机盐类也可用于填充剂,包括三磷酸钙,碳酸镁和氯化钠。 Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. 一些可通过商业渠道获得的稀释剂包括Fast-Flo,Emdex,STA-Rx 1500,Emcompress和Avicell。 Some commercially available diluents channels, including Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

治疗性药物制剂中还应包括崩解剂以构成固体剂型。 Therapeutic pharmaceutical formulation should also include a disintegrant to form a solid dosage form. 用作崩解剂的物质包括但不限于淀粉,包括基于淀粉的商业性崩解剂Explotab。 Substances used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. 淀粉羟乙酸钠,安伯莱特,羧甲基纤维素钠,ultramylopectin,藻酸钠,白明胶,桔皮,酸性羧甲基纤维素,天然海绵和斑脱土也都可以应用。 Sodium starch glycolate, Amber Wright, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite can also be used. 崩解剂的另一种形式是不溶性阳离子交换树脂。 Another form of the disintegrants are the insoluble cationic exchange resins. 粉状树脂也可用作崩解剂和粘合剂,包括粉状树脂如琼脂,刺梧桐树胶或黄芪胶。 Powdered resin may be used as disintegrants and as binders, including powdered resin such as agar, Karaya or tragacanth. 褐藻酸及其钠盐也可作为崩解剂。 Alginic acid and its sodium salt may also be used as a disintegrant.

粘合剂可以将治疗制剂结合在一起,形成坚硬的片剂。 The binder may be combined therapeutic formulation to form a hard tablet. 粘合剂包括来自天然产物的物质,如阿拉伯胶,黄芪胶,淀粉和白明胶。 Binders include materials from natural products such as acacia, tragacanth, starch and gelatin. 其他包括甲基纤维素(MC),乙基纤维素(EC),以及羧甲基纤维素(CMC)。 Others include methyl cellulose (MC), ethyl cellulose (EC), carboxymethyl cellulose (CMC). 聚乙烯吡咯烷(PVP)和羟丙甲基纤维素(HPMC)均可用于乙醇溶液以形成治疗性药物颗粒。 Polyvinylpyrrolidine (PVP) and hydroxypropylmethyl cellulose (HPMC) may be used in an ethanol solution to form particles of a therapeutic drug.

治疗性药物制剂中还可包括一种抗摩擦剂以防止在制备过程中的粘连。 Therapeutic pharmaceutical formulation may further include one anti-friction agent to prevent adhesion in the production process. 在治疗性药物和死壁之间可以应用润滑剂作为分层,这些润滑剂包括但不限于硬脂酸,其中包括其镁盐和钠盐,聚四氟乙烯(PTFE),液体石蜡,植物油和蜡。 Therapeutic drugs and between the die wall lubricant can be used as a stratification, but are not limited to such lubricants include stearic acid, including its magnesium and sodium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and wax. 可溶性润滑剂也可应用,如月桂基硫酸钠,月桂基硫酸镁,多种分子量的聚乙二醇,Carbowax 4000和6000。 Soluble lubricants may also be applied, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

可以添加在制备过程中能够改善药物流体特性、在压缩过程中能够帮助再排列的glidants。 May be added during the manufacturing process can be improved pharmaceutical properties of the fluid, the compression process can help rearranged glidants. Glidants可以包括淀粉,滑石,火成硅,水合硅铝酸盐。 Glidants may include starch, talc, fumed silica, hydrated aluminosilicate.

为了帮助治疗性药物在液体环境中解散,可以添加表面活性剂作为湿润剂。 To help therapeutic drug dissolved in a liquid environment, a surfactant may be added as a wetting agent. 表面活性剂可包括阴离子去污剂,如月桂基硫酸钠,二辛基磺基琥珀酸钠和二辛磺酸钠。 Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. 阳离子去污剂也可应用,包括氯苄烷铵或氯苄乙铵。 Cationic detergent may be applied, include benzalkonium chloride or benzethonium chloride. 在制备过程中可作为表面活性剂的潜在非离子去污剂为lauromacrogol 400,聚乙二醇40硬脂酸酯,聚氧乙烯加氢蓖麻油10,50和60,甘油单硬脂酸酯,聚山梨醇酯40,60,65和80,蔗糖脂肪酸酯,甲基纤维素和羧甲基纤维素。 In the preparation process as a potential non-ionic detergent surfactant is lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40,60,65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. 这些表面活性剂可以单独或以不同比例以混合物的形式存在于蛋白或衍生物制剂中。 These surfactants may be used alone or as a mixture in different ratios of protein or derivative thereof present in the formulation.

具有增加化合物摄取潜力的添加剂如脂肪酸十八烯酸,亚油酸和亚麻酸。 Additives having increased potential of a compound such as fatty acid uptake octadecenoic acid, linoleic acid and linolenic acid.

控释制剂是备受期待的。 Controlled-release formulation is highly anticipated. 药物可以与惰性基质整合,该惰性基质允许以扩散或滤过的机制,如树胶释放。 Integration may be an inert pharmaceutical substrate, the inert matrix permits the diffusion or filtration mechanisms, such as gums release. 缓慢降解的基质如藻酸盐,多糖也可整合入制剂。 Slowly degrading matrix such as alginate, polysaccharides can also be integrated into the formulation. 该治疗性药物的另一种可控释放是通过基于Oros治疗系统(Alza股份有限公司)的方法,即将药物密封在半透膜中,该半透膜允许水分子进入,并在渗透效应作用下将药物通过唯一一个小型开口挤出。 The therapeutic drugs Another controlled release is obtained by a method based Oros therapeutic system (Alza Corp.), and about to seal the drug in the semi-permeable membrane, the semi-permeable membrane allows water molecules to enter and role in osmotic effect the drug is extruded through a single small opening. 一些肠衣制剂也有缓释效应。 Some casings sustained release formulations have effect.

其他包膜也可用于制剂。 Other capsule formulation may also be used. 这些包膜包括各种可以制成糖衣的糖类。 These include a variety of envelope may be made of icing sugar. 治疗性制剂还可以薄膜封衣的片剂给出,用于该情况的物质可分为2类。 Therapeutic formulations may also seal the film coated tablets are given for the case where the material can be divided into two categories. 第一类是非肠衣物质,包括甲基纤维素,乙基纤维素,羟乙基纤维素,甲羟乙基纤维素,羟丙基纤维素,羟丙甲基纤维素,羧甲基纤维素钠,providone和聚乙二醇。 The first class of non-enteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methyl hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose , providone and polyethylene glycol. 第二类包括常见的邻苯二甲酸酯的肠衣类物质。 The second category comprises common enteric phthalate species.

可用混合物提供最适封衣包膜。 Available sealing coating mixture to provide optimum envelope. 封衣包膜可在平锅式封衣机或流化床或通过压缩封衣的方式实施。 Seal coat can be coated pan coating machine or a fluidized bed type seal or seal compression by way of embodiments in clothing.

本发明蛋白质(或其衍生物)通过肺给药也在考虑之列。 Protein (or derivatives thereof) of the present invention is by pulmonary administration are also contemplated. 所述蛋白质(或衍生物)通过吸入进入哺乳动物的肺脏,并穿过肺上皮细胞层进入血液。 The protein (or derivative) to a mammal by inhalation into the lungs, and traverses across the lung epithelial cell layer into the blood. (有关于此的其他报道包括Adjei等,药理学研究,7:565-569(1990);Adjei等,药理学国际杂志,63:135-144(1990)(醋酸leuprolide);Braquet等,心血管药理学杂志,13(Suppl.5):s.143-146(1989)(内皮素-1);Hubbard等,内科学纪事,3:206-212(1989)(α1-抗胰蛋白酶);Smith等,临床研究杂志,84:1145-1146(1989)(α1-蛋白酶);Oswein等,“蛋白质的气雾化”,呼吸系统药物给药研讨会II,Keystone,美国科罗拉多州,1990年3月(重组人生长激素);Debs等,免疫学杂志,140:3482-3488(1988)(γ-干扰素和α-肿瘤坏死因子);以及Platz等,美国专利5 284 656(粒细胞集落刺激因子))。 (Other reports of this include Adjei et al., Pharmacological research, 7: 565-569 (1990); Adjei et al., International Journal of Pharmaceutical Science, 63: 135-144 (1990) (leuprolide acetate); Braquet, etc., cardiovascular Journal of Pharmacology, 13 (Suppl.5): s.143-146 (1989) (endothelin -1); Hubbard et al., Annals of internal medicine, 3: 206-212 (1989) (α1- antitrypsin); Smith and so on, Journal of clinical Research, 84: 1145-1146 (1989) (α1- proteinase); Oswein et al., "aerosolization of protein", respiratory system drugs administered seminar II, Keystone, Colorado, USA, March 1990 (recombinant human growth hormone); Debs et al., J. Immunol., 140: 3482-3488 (1988) (γ- α- interferon and TNF); and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor )).

本发明的实际应用可考虑为治疗性产品肺部给药而设计的范围宽泛的器械装置,包括但不限于喷雾器,定量吸入器,和干粉式吸入器,本领域技术人员对所有这些装置都是熟悉的。 Practice of the invention may be considered a wide range of devices for pulmonary administration apparatus is designed for therapeutic products, including but not limited to nebulizers, metered dose inhalers, and dry powder inhalers, the present art for the art all of which are familiar.

一些适用于本发明实际应用并可通过商业途径获得的特定装置实例如Ultravent喷雾器,由位于密苏里州圣路易斯的Mallinckrodt有限公司生产;Acorn II喷雾器,由位于科罗拉多州Englewood的Marquest医学产品公司生产;Ventolin定量吸入器,由位于北卡罗来纳州研究三角公园的葛兰素股份有限公司生产;以及Spinhaler干粉式吸入器,由位于马赛诸塞州Bedford的Fiscons有限公司生产。 Some suitable to practice the present invention and a specific example of an apparatus such as a commercially available Ultravent nebulizer by Mallinckrodt of St. Louis, Missouri Co; Acorn II nebulizer, manufactured by Englewood, Colorado Marquest Medical Products Company; the Ventolin quantitative inhalers, by the North Carolina Research triangle Park, GlaxoSmithKline, Ltd. manufactures; Spinhaler and dry powder inhalers, located by the Massachusetts Bedford, Fiscons Co.

所有这些装置都要求制剂配方的应用要适宜于本发明化合物的喷发。 All of these devices require the application of the formulation to formulations suitable for the compounds of the present invention eruption. 典型的,每种制剂配方特定于一种使用的装置,并在稀释剂、佐剂和/或载体外,包括应用一种在治疗中非常有用的适宜的推进剂。 Typically, each formulation recipe specific apparatus for use, and an outer diluents, adjuvants and / or carriers, comprises a very useful application in the treatment of a suitable propellant.

最具优势的本发明化合物是制备成平均颗粒大小小于10μm(或微米)的特定形式,最优选0.5-5μm,因为可以最有效的到达肺脏末梢。 The most advantageous compounds of the present invention is a specific form having an average particle size of less than 10 m (or microns) is prepared, most preferably 0.5 to 5 m, because the lungs can be most effectively reaches the tip.

载体包括碳水化合物,如海藻糖,甘露醇,木糖醇,蔗糖,乳糖和山梨醇。 Carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. 其他配方中应用的成分可包括DPPC,DOPE,DSPC和DOPC。 Other formulations use ingredients may include DPPC, DOPE, DSPC and DOPC. 天然或合成的表面活性剂也可应用。 Natural or synthetic surfactants may be used. 还可应用聚乙二醇(甚或除了应用于衍生蛋白质或类似物)。 Polyethylene glycol may also be applied (or even applied except derived proteins or the like). 葡聚糖,如环化葡聚糖也可应用。 Dextran, such as dextran ring can also be applied. 胆酸盐和其他的相关增强剂也可应用。 Bile salts and other related enhancers may be used. 纤维素和纤维素衍生物也可应用。 Cellulose and cellulose derivatives may be used. 氨基酸也可应用,如用于缓冲配方。 Amino acids may also be applied, such as for buffering the formulation.

同样,应用脂质体,微胶囊,微球,复杂联合体或其他类型的载体也在考虑之列。 Similarly, the use of liposomes, microcapsules, microspheres, complex or other complex types of carriers is also contemplated.

适用于喷雾器,无论喷射器或超声喷雾器,的典型配方制剂包括将本发明化合物以大约每毫升溶液含0.1-0.25mg生物活性蛋白的浓度溶于水。 Suitable nebulizer, either jet or ultrasonic nebulizer, the formulation typical formulation comprises a compound of the present invention at a concentration of about 0.1-0.25mg per ml of solution containing water-soluble biologically active proteins. 配方还可包括一种缓冲液和单糖(如为蛋白质稳定性和渗透压调节)。 Formulations may further include one buffer and a simple sugar (such as the regulation of protein stability and osmotic pressure). 喷雾器配方还可包括一种表面活性剂,以减少或防止在气雾形成过程中溶液气化导致的表面诱导性蛋白聚合。 Nebulizer formulation may further comprise a surfactant, to reduce or prevent surface-induced protein solution is vaporized during aerosol formation caused polymerization.

用于定量吸入器装置的配方通常包括借助于表面活性剂而悬浮于推进剂中的含有本发明化合物的细小粉粒。 Formulation for metered dose inhaler device generally comprise a propellant and suspended in fine particles containing a compound of the invention by means of a surfactant. 推进剂可以是适用于该目的的任一常规物质,如含氯氟烃,氢氯氟烃,烃氟烃,或烃,包括三氯氟甲烷,二氯二氟甲烷,二氯四氟乙醇,以及1,1,1,2-四氟乙烷,或其组合物。 Propellant may be any conventional material suitable for this purpose, such as chlorofluorocarbon, hydrochlorofluorocarbon, fluorocarbon hydrocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethane ethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. 适宜的表面活性剂包括山梨醇三油酸酯和大豆卵磷脂。 Suitable surfactants include sorbitan trioleate and soya lecithin. 十八烯酸也可作为表面活性剂应用。 Oleic acid as a surfactant can also be applied.

从干粉式吸入器装置中喷发的配方制剂应包括含有本发明化合物的细小干粉颗粒,还可包括一定量的膨胀剂,如乳糖,山梨醇,蔗糖,甘露醇,海藻糖或木糖醇,如50-90%配方重量,这些膨胀剂使干粉从装置中的喷发更加容易。 Erupted from the dry powder inhaler device comprising a formulation of fine dry powder particles of the formulation should contain the compound of the present invention may further comprise an amount of a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol, as 50-90% by weight of the formulation, these dry powder bulking agent from the device easier eruption.

本发明化合物的鼻腔给药也在考虑之列。 Nasal administration the compounds of the present invention are also contemplated. 鼻腔给药允许鼻腔给予治疗性药物后,蛋白质直接进入血液,产品无需在肺部沉积。 After nasal administration to allow intranasal administration of therapeutic drugs, proteins directly into the blood products without deposition in the lungs. 鼻腔给药的配方包括含有葡聚糖或环状葡聚糖的药物。 Formulations for nasal administration comprising a drug containing a cyclic dextran or dextran. 通过其他粘膜运输给药的方式也在考虑之列。 Other ways are also contemplated by transmucosal administration transportation columns.

剂量治疗上述病况方法的剂量方案需由主治医师综合考虑多种影响药物作用的因素后决定,影响因素如患者的年龄、病情、体重、性别和饮食情况,任何感染的严重性,给药时间和其他临床因素。 After dosing regimen dose treatment methods require the above conditions by the attending physician considering various factors that affect drug action decisions, factors such as the patient's age, condition, weight, sex, and diet, the severity of any infection, time of administration and other clinical factors. 通常剂量范围为每公斤体重每天0.1μg-100mg本发明化合物,优选0.1-1000μg/kg;更优选0.1-150μg/kg,以每天剂量给予,或以相当剂量以更长或更短的间隔给予,如隔天给药,每周两次给药,每周一次给药,或每日两次或三次给药。 Usual dose range per kilogram of body weight per day of a compound of the present invention is 0.1μg-100mg, preferably 0.1-1000μg / kg; more preferably 0.1-150μg / kg, in daily doses or in equivalent doses at longer or shorter intervals administered, The next day administration, twice-weekly dosing, administered weekly, or twice or three times a day administration.

本发明化合物可通过首剂弹丸注射后持续输注来维持循环中药物的治疗水平。 The compounds of this invention may be continuous infusion to maintain therapeutic circulating levels of drug through the first dose bolus injection. 如另一例所示,本发明化合物也可以一次剂量给药。 As another example, the compound of the present invention may also be administered in a single dose. 这些本领域常规技术应通过优良的医学实践结合患者个体的临床情况,决定最优化有效剂量和给药方案。 These conventional in the art should be combined with the clinical condition of the individual patient, by good medical practice, the decision to optimize effective dosages and administration regimen. 给药频率有赖于药物的药代动力学参数和给药途径。 The frequency of administration and the pharmacokinetic parameters of the drug depend on the route of administration. 最佳药物配方应由本领域技术人员根据给药途径和期望剂量决定。 Optimal pharmaceutical formulation should be decided according to the person skilled in the route of administration and desired dosage. 见,例如,雷明顿药理学,第18版(1990,Mack出版有限公司,Easton,巴拿马,18042),1435-1712页,此书在此引入,作为参考。 See, for example, Remington Pharmacology, 18th Edition (1990, Mack Publishing Co., Easton, Panama, 18042), pages 1435-1712, the book is hereby incorporated by reference. 这些配方会影响药物的物理特性,稳定性,体内释放率和体内清除率。 These formulations can affect the physical properties of the drug, stability, in vivo release rate of in vivo clearance. 根据给药途径,适宜的剂量可以根据体重,体表面积或器官大小计算得出。 The route of administration, a suitable dose may be calculated according to the derived weight, body surface area or organ size. 为决定包括每一种上述配方的治疗适宜剂量而必须进行的进一步精细计算通常是由本领域技术人员在适宜的试验条件下给出的,特别是根据这里公开发表的剂量信息和试验方法,以及上述在人类临床试验中观察到的药代动力学数据。 To determine further comprises calculating a fine therapeutic dose must be suitable for each of the above formulations are generally given by those skilled in the art under suitable experimental conditions, especially in light of the dosage information and published test methods herein, and the above We observed in clinical trials of human pharmacokinetic data. 适宜的剂量可以通过应用业已建立的测定血液剂量水平方法结合适宜的剂量-反应资料而得到确认。 Information obtained by the reaction was confirmed - a suitable dose may be combined by determining a suitable dose blood dosage level Methods already established. 最终剂量方案应由主治医师综合考虑多种可能影响药物作用的因素决定,如药物的具体活性,损伤的严重程度和患者的反应情况,患者的年龄,病情,体重,性别和饮食情况,任何感染的严重性,给药时间以及其他临床因素。 The final dosage regimen by the attending physician considering various factors that may affect drug action decisions, such as the specific activity of the drug, reactions of the severity of the injury and the patient, the patient's age, condition, weight, sex and diet, any infection severity, time of administration and other clinical factors. 随着研究的进行,针对多种疾病和病况的有关适宜剂量水平和治疗时间的进一步信息将不断产生。 As studies are conducted, further information regarding the appropriate dosage levels and duration of treatment for various diseases and conditions will continue to produce.

本发明的治疗方法、组合物和化合物可单独或与其他细胞因子、可溶性Mpl受体、造血因子、白细胞介素、生长因子或抗体联合,在以其他症状合并血小板减少为特征的疾病状态的治疗中使用。 The method of treatment of the present invention, compositions and compounds can be used alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors or antibodies in combination, characterized by the treatment in reducing the symptoms of other disease states combined platelet use. 预计本发明化合物在与一般造血刺激因子如IL-3或GM-CSF联合使用,治疗某些类型的血小板缺乏症时,将显示疗效。 Expected when compounds of the present invention with the general hematopoietic stimulating factors such as IL-3 or GM-CSF used in combination, the treatment of certain types of thrombocytopenia, the display effect. 其他巨核细胞刺激因子,如meg-CSF,干细胞因子(SCF),白血病抑制因子(LIF),制瘤素M(OSM),或其他具有巨核细胞刺激活性的分子也可与Mpl配体合用。 Other megakaryocyte stimulating factor, such as meg-CSF, stem cell factor (SCF), leukemia inhibitory factor (of LIF), oncostatin M (OSM), or other with megakaryocyte stimulating activity of the molecule may also be used in combination with Mpl ligand. 其他可以如此联合给药的细胞因子或造血因子的实例包括,IL-1α,IL-1β,IL-2,IL-3,IL-4,IL-5,IL-6,IL-11,集落刺激因子-1(CSF-1),M-CSF,SCF,GM-CSF,粒细胞集落刺激因子(G-CSF),EPO,α-干扰素(IFN-α),同型干扰素,IFN-β,IFN-γ,IL-7,IL-8,IL-9,IL-10,IL-12,IL-13,IL-14,IL-15,IL-16,IL-17,IL-18,血小板生成素(TPO),血管生成素(angiopoietins),例如Ang-1,Ang-2,Ang-4,Ang-Y,人血管生成素样蛋白,血管内皮生长因子(VEGF),血管生成素(angiogenin),骨形态发生蛋白-1,骨形态发生蛋白-2,骨形态发生蛋白-3,骨形态发生蛋白-4,骨形态发生蛋白-5,骨形态发生蛋白-6,骨形态发生蛋白-7,骨形态发生蛋白-8,骨形态发生蛋白-9,骨形态发生蛋白-10,骨形态发生蛋白-11,骨形态发生蛋白-12,骨形态发生蛋白-13,骨形态发生蛋白-14,骨形态发生蛋白-15,骨形态发生蛋白受体IA,骨 Other possible examples of such a joint cytokines or hematopoietic factors for administration include, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor -1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, α- interferon (IFN-α), with interferon, IFN-β, IFN-γ, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, thrombopoietin Su (TPO), angiogenin (Angiopoietins), for example Ang-1, Ang-2, Ang-4, Ang-Y, human angiopoietin-like protein, vascular endothelial growth factor (VEGF), angiopoietin (of angiogenin in) , bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, -5 bone morphogenic proteins, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein -10, -11 bone morphogenic proteins, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein -14, bone -15 morphogenic proteins, bone morphogenic protein receptor IA, bone 形态发生蛋白受体IB,脑衍生神经营养因子,纤毛神经营养因子,纤毛神经营养因子受体α,细胞因子诱导的神经营养趋化因子1,细胞因子诱导的神经营养趋化因子2α,细胞因子诱导的嗜中性粒细胞趋化因子1,细胞因子诱导的嗜中性粒细胞趋化因子2α,细胞因子诱导的嗜中性粒细胞趋化因子2β,β内皮细胞生长因子,内皮素1,内皮生长因子,内皮衍生嗜中性粒细胞趋化剂(attractant),成纤维细胞生长因子4,成纤维细胞生长因子5,成纤维细胞生长因子6,成纤维细胞生长因子7,成纤维细胞生长因子8,成纤维细胞生长因子8b,成纤维细胞生长因子8c,成纤维细胞生长因子9,成纤维细胞生长因子10,酸性成纤维细胞生长因子,碱性成纤维细胞生长因子,神经胶质细胞系衍生神经营养因子受体α1,神经胶质细胞系衍生神经营养因子受体α2,生长相关性蛋白, The IB morphogenetic protein receptor, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor [alpha], cytokine-induced neurotrophic chemokine 1, cytokine-induced neurotrophic factor 2 [alpha chemokines, cytokines induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2 [alpha, cytokine-induced neutrophil chemotactic factor 2β, β endothelial cell growth factor, endothelin 1, endothelial growth factor, endothelial cell-derived neutrophil chemotactic agent (Attractant), fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8B, 8C fibroblast growth factor, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acidic, basic fibroblast growth factor, glial cells line derived neurotrophic factor receptor alpha] l, glial cell line-derived neurotrophic factor receptor alpha] 2, growth related protein, 长相关性蛋白α,生长相关性蛋白β,生长相关性蛋白γ,肝素结合性上皮生长因子,肝细胞生长因子,肝细胞生长因子受体,胰岛素样生长因子I,胰岛素样生长因子受体,胰岛素样生长因子II,胰岛素样生长因子结合蛋白,角化细胞生长因子,白血病抑制因子,白血病抑制因子受体α,神经生长因子,神经生长因子受体,神经营养素-3,神经营养素-4,胎盘生长因子,胎盘生长因子2,血小板衍生内皮细胞生长因子,血小板衍生生长因子,血小板衍生生长因子A链,血小板衍生生长因子AA,血小板衍生生长因子AB,血小板衍生生长因子B链,血小板衍生生长因子BB,血小板衍生生长因子受体α,血小板衍生生长因子受体β,前B细胞生长刺激因子,干细胞因子受体,TNF,包括TNFO,TNF1,TNF2,转化生长因子α,转化生长因子β,转化生长因子β1,转化生长因子β Long-related protein [alpha], growth related protein beta], growth associated protein gamma], heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor [alpha], nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placental growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor [alpha], platelet-derived growth factor receptor beta], pre-B cell growth stimulating factor, stem cell factor receptor, TNF, including TNFO, TNF1, TNF2, transforming growth factor [alpha], transforming growth factor beta], transforming growth factor β1, transforming growth factor β 1.2,转化生长因子β2,转化生长因子β3,转化生长因子β5,潜在转化生长因子β1,转化生长因子β结合蛋白I,转化生长因子β结合蛋白II,转化生长因子β结合蛋白III,I型肿瘤坏死因子受体,II型肿瘤坏死因子受体,尿激酶型血浆纤维蛋白溶酶原激活物受体,血管内皮生长因子,以及嵌合蛋白及其具有生物学或免疫学活性的部分。 1.2, transforming growth factor beta] 2, transforming growth factor beta] 3, transforming growth factor-beta5, latent transforming growth factor-beta1, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, type I tumor necrosis factor receptor, tumor necrosis factor receptor type II, urokinase-type plasma plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active portions. 同时或依次给予有效剂量的可溶性哺乳动物Mpl受体对给药者来说也是有用的,该受体具有巨核细胞一旦成熟,就使巨核细胞分裂为血小板的效用。 Simultaneously or sequentially administering an effective amount of a soluble mammalian Mpl receptor is also useful for administration are concerned, the receptor once mature megakaryocyte, megakaryocyte causes the split utility platelets. 因此,给予本发明化合物(增加成熟巨核细胞数量)后,再给予可溶性Mpl受体(灭活配体并允许成熟巨核细胞产生血小板),对于刺激血小板产生是一个具有特别预期效果的方法。 Thus, after administration of the compound of the present invention (to increase the number of mature megakaryocytes), and then administering the soluble Mpl receptor (to inactivate the ligand and allow the mature megakaryocytes to produce platelets), for stimulating production of platelets having the desired effect it is a particular method. 上述剂量可以调整以补偿在治疗性组合物中添加的成分。 The above dosage may be adjusted to compensate for the added components in the therapeutic composition. 患者的治疗进展可通过常规方法进行监控。 Advances in the treatment of patients can be monitored by conventional methods.

在将本发明化合物加入血小板和/或巨核细胞以及相关细胞组合物中时,包括的数量通常可用本领域熟知的技术和方法试验确认。 When the compound of the present invention added platelets and / or megakaryocytes and related cells in the composition, including the number of available techniques and methods generally known in the art confirmation tests. 示例数量范围是每106个细胞加入本发明化合物0.1μg-1mg。 Example of the number range per 106 cells was added the compound of the present invention is 0.1μg-1mg.

应当理解,将本发明的教导应用于特定的问题或情况时,应根据这里给出的教导,并在本领域技术人员的能力范围内。 It should be understood that the teachings of the teachings of the present invention is applied to a specific problem or situation, it should be given here in accordance with, and within the scope of those skilled in the art. 下面将给出本发明产品及其代表性分离、应用及生产方法。 The present invention will be given below and the product isolated representation, application and production process.

实施例I.下面将介绍这里公开的一些第一组化合物的制备方法示例。 EXAMPLES I. The following example describes the preparation of some of the first group of compounds disclosed herein.

A.材料与方法所有用于肽合成的氨基酸衍生物(全部是L构型)和树脂均购于Novabiochem公司。 A. Materials and Methods All amino acid derivatives (all of L configuration) and resins used in peptide synthesis were purchased from Novabiochem. 肽合成试剂(DCC,HOBt等)以溶液形式购于应用生物系统股份有限公司。 Peptide synthesis reagents (DCC, HOBt, etc.) were purchased in the form of a solution in an Applied Biosystems Corporation. 两种PEG衍生物购于Shearwater Polymers股份有限公司。 Two kinds of PEG derivatives available from Shearwater Polymers Limited. 所有的溶剂(二氯甲烷,N-甲基吡咯烷酮,甲醇,乙腈)购于EM Sciences公司。 All solvents (dichloromethane, N- methylpyrrolidinone, methanol, acetonitrile) were purchased from EM Sciences companies. 在Beckman系统的Vydac柱(0.46cm×25cm,C18反相,5mm)上进行HPLC分析,洗脱流速为1ml/分,并在220和280nm处进行双重UV探测。 (, C18 reverse phase, 0.46cm × 25cm 5mm) column Vydac performed on a Beckman HPLC system analysis, eluting with a flow rate of 1ml / min, and UV detection at 220 and double at 280nm. 在所有HPLC操作中应用线形梯度的两个移动相,缓冲液A-水(0.1%TFA)和缓冲液B-乙腈(0.1%TFA)。 Linear gradient of two mobile application in all operating phase HPLC, Buffer A- Water (0.1% TFA) and Buffer B- acetonitrile (0.1% TFA). 这里引用的编号肽,如17b,18,19和20,可参考表1的数字命名,其中一些在图2和3中有进一步的图示。 No peptides herein by reference, such as 17b, 18,19 and 20, reference numeral 1 Table name, some of which are further illustrated in FIGS. 2 and 3.

肽合成。 Peptide synthesis. 所有的肽都通过已经建立的成熟的分步式固相合成法制备。 All peptides were prepared by mature stepwise solid phase synthesis method has been established. 运用Fmoc化学的固相合成法在ABI肽合成仪上进行。 Using Fmoc chemistry for solid phase synthesis on an ABI peptide synthesizer. 典型的,肽合成的开始是预装载0.1mmol的Wang树脂。 Typically, peptide synthesis began as a preloaded Wang resin 0.1mmol. Fmoc去保护按标准哌啶法进行。 Fmoc deprotection carried out according to standard methods piperidine. 用DCC/HOBt实施偶联。 With DCC / HOBt coupling embodiment. 侧链保护基团是:Glu(Ot-Bu),Thr(t-Bu),Arg(Pbf),Gln(Trt),Trp(t-Boc)和Cys(Trt)。 Side chain protecting groups are: Glu (Ot-Bu), Thr (t-Bu), Arg (Pbf), Gln (Trt), Trp (t-Boc) and Cys (Trt). 第--个肽前体聚乙二醇化时,Dde用来侧链保护接头上的赖氨酸,Boc-Ile-OH用作最后一个偶联。 - of time before the pegylated peptides thereof, Dde side-chain protection for lysine linker, Boc-Ile-OH used as the last coupling. 通过应用无水肼(2%溶于NMP,3×2min)去除Dde,然后在DCC的作用下用溴乙酸酐进行偶联。 Dde is removed by application of anhydrous hydrazine (2% dissolved in NMP, 3 × 2min), followed by coupling with bromoacetic anhydride under the action of DCC. 对肽18来说,位于接头上的半胱氨酸侧链通过三苯甲基进行保护。 For peptide 18, the cysteine ​​side chains located on the joint is protected by a trityl group. 最后的去保护和所有肽-树脂的断裂在RT(室温)下实施4小时,应用含2.5%水,5%苯酚,2.5%三异丙基硅烷和2.5%巯基苯甲醚的三氟乙酸(TFA)。 The final deprotection and all peptide - resin is broken for 4 hours at RT (room temperature), application of water containing 2.5%, 5% phenol, 2.5% triisopropylsilane and 2.5% trifluoroacetic acid mercapto anisole ( TFA). 去除TFA后,断裂肽用冷的无水乙醚沉淀。 After removal of TFA, the peptide precipitated with cold break anhydrous ethyl ether. 应用原材料在溶于水的15%DMSO(pH 7.5)中直接进行环状肽二硫键的构建。 Application of water-soluble material in 15% DMSO cyclic peptide construct directly disulfide bond (pH 7.5) in. 所有的原始肽都通过制备性反向HPLC纯化,并通过ESI-MS和氨基酸分析确认肽结构。 All original peptides were purified by preparative reverse-phase HPLC, and analyzed for the peptide structure confirmed by ESI-MS and amino acids.

另外,上述所有肽还可以应用t-Boc化学法制备。 Further, all of the above peptides may also be prepared by application of t-Boc chemistry method. 在这种情况下,起始树脂是经典的Merrifield或Pam树脂,侧链保护基团是:Glu(OBzl),Thr(Bzl),Arg(Tos),Trp(CHO),Cys(p-MeBzl)。 In this case, the starting resin is the classic Merrifield or Pam resin, and side chain protecting groups are: Glu (OBzl), Thr (Bzl), Arg (Tos), Trp (CHO), Cys (p-MeBzl) . 可应用氢氟酸(HF)最后断裂肽树脂。 Application may be hydrofluoric acid (HF) and finally break the peptide resin.

本研究所述所有含有天然氨基酸接头的串联二聚体肽也可通过重组DNA技术制备。 In this study all the natural amino acids containing a tandem dimer peptide linker can also be prepared by recombinant DNA techniques.

聚乙二醇化业已发展的一种合成肽聚乙二醇化的新型聚汇策略包括通过在溶液中形成结合连接,使肽和聚乙二醇(PEG)部分结合,所述肽和PEG各携带一个可以相互反应的具体官能团。 A synthetic peptide pegylated pegylated have been developed for a novel strategy Juhui formed by bonding connection comprising in solution, a peptide and a polyethylene glycol (PEG) binding portion of the peptide, and each carrying a PEG specific functional groups may react with each other. 前体肽可容易地通过上述常规的固相合成法制备。 Precursor peptides can be easily prepared by conventional solid-phase synthesis described above. 如下所述,这些肽在特定位点携带适宜官能团,并处于“预激活”状态。 As described below, these peptides carry a suitable functional group at a particular site, and in the "pre-activated" state. 这些前体在与PEG部分反应前经过纯化并进行充分的鉴定。 The precursor is purified prior to reaction with the PEG moiety and fully identified. 肽和PEG的连接通常在液相发生,并可通过反向分析性HPLC容易地监控。 And attachment of PEG to peptides normally occurs in the liquid phase and can be easily monitored by reverse analytical HPLC. 聚乙二醇化的肽可通过制备性HPLC容易地得到纯化,并通过分析性HPLC、氨基酸分析和激光解吸质谱法确认。 PEGylated peptides can be easily purified by preparative HPLC, and by analytical HPLC, amino acid analysis and laser desorption mass spectrometry confirmed.

肽19的制备。 Preparation 19 peptide. 将肽17b(12mg)和MeO-PEG-SH 5000(30mg,2等份)溶解于1ml含水缓冲液中(pH8)。 Peptide 17b (12mg) and MeO-PEG-SH 5000 (30mg, 2 aliquots) was dissolved in 1ml aqueous buffer (pH8). 混合物室温下孵育30分钟,应用分析性HPLC检测反应,并显示反应完成80%以上。 The mixture was incubated at room temperature for 30 minutes, and applied analytical HPLC detection reaction, and the reaction was complete by 80% or more. 聚乙二醇化的物质通过制备性HPLC分离。 Pegylated material was separated by preparative HPLC.

肽20的制备。 Preparation of peptide 20. 将肽18(14mg)和MeO-PEG-马来酰亚胺(25mg)溶解于1.5ml缓冲液中(pH8)。 The peptide 18 (14mg) and MeO-PEG- maleimide (25mg) was dissolved in 1.5ml buffer (pH8). 混合物室温下孵育30分钟,此时,通过将一部分样本加到HPLC柱上,用分析性HPLC监控发现,完成~70%的转化。 The mixture was incubated for 30 minutes at room temperature, at this time, part of the sample by adding the HPLC column, monitored by analytical HPLC was found to complete the conversion to 70%. 聚乙二醇化的物质通过制备性HPLC分离。 Pegylated material was separated by preparative HPLC.

生物活性检测法。 Biological activity assay. TPO的体外生物活性检测是通过有丝分裂法检测的,该方法应用转染了人mp1受体的IL-3依赖型鼠32D克隆细胞。 TPO in vitro bioassay is a mitogenic by detecting method, the method is applied transfected with murine IL-3 dependent 32D cell clone human mp1 receptor. 这一方法在WO95/26746中有详尽描述。 This method is described in detail in WO95 / 26746. 细胞在含有10%胎克隆II和1ng/ml mIL-3的MEM培养基中维持。 Cells containing 10% fetal Clone II and 1ng / ml MEM medium maintained in mIL-3. 在加入样本前,细胞用缺乏mIL-3的生长培养基洗脱两次。 Before samples were added, cells were treated with growth medium lacking mIL-3 was eluted twice. 准备延伸十二点的TPO标准曲线,范围为3333-39pg/ml。 Preparation extending twelve o'clock TPO standard curve ranging 3333-39pg / ml. 每种样品制备四种预计位于标准曲线线性部分(1000-125pg/ml)的稀释液,并做三个重复。 Four kinds of samples were prepared for each dilution is expected in the standard curve which (1000-125pg / ml), and to make three repeats. 在每孔含有10000个细胞的96孔板的适宜孔中加入100μl每种稀释度的样品或标准品。 Add 100μl of each dilution of sample or standard 96-well plate in a suitable hole in each well containing 10,000 cells. 在37C,10%CO2条件下孵育44小时后,每孔加入MTS(一种可被细胞生物还原为甲的四唑化合物)。 After incubation at 37C, 10% CO2 conditions for 44 hours, the MTS was added per well (one kind of biological cells can be reduced to A  tetrazole). 大约6小时后,在490nm下读取每孔的光密度。 After about six hours, the optical density of each well was read at 490nm. 剂量反应曲线(TPO浓度的对数值比背景光密度值)产生,并可对位于标准曲线线性部分的点进行线性回归分析。 Dose response curves (TPO concentration values ​​of optical density than the background) is generated, and can be located in the linear portion of the standard curve points linear regression analysis. 未知测试样品的浓度可通过获得的线性方程式和校正稀释因子而得到。 The concentration of the unknown test samples can be obtained by a linear equation and a correction obtained by the dilution factor.

缩略语。 Abbreviations. HPLC:高效液相层析;ESI-MS:电子束离子化质谱测定法;MALDI-MS:基质协助的激光解吸离子化质谱测定法;PEG:聚(乙二醇)。 HPLC: high performance liquid chromatography; ESI-MS: electron-beam ionization mass spectrometry; MALDI-MS: Matrix-assisted laser desorption ionization mass spectrometry; PEG: Poly (ethylene glycol). 所有的氨基酸都以标准3个字母或1个字母编码表示。 All amino acids are represented by standard three letter or one letter code. T-Boc:叔丁氧羰基;tBu:叔丁基;Bzl:苯甲基;DDC:二环己基碳二亚胺;HOBt:1-羟苯三唑;NMP:N-甲基-2-吡咯烷酮;Pbf:2,2,4,6,7-五甲基二氢-苯并呋喃-5-磺酰;Trt:三苯甲基;Dde:1-(4,4-二甲基-2,6-双氧-亚环己基)乙烷。 T-Boc: t-butoxycarbonyl group; tBu: t-butyl; Bzl: benzyl; DDC: dicyclohexyl carbodiimide; HOBt: 1- hydroxyphenyl benzotriazole; NMP: N- methyl-2-pyrrolidone ; Pbf: 2,2,4,6,7- pentamethyl-dihydro - benzofuran-5-sulfonyl; Trt: trityl; Dde: 1- (4,4- dimethyl-2, 6- dioxygen - cyclohexylidene) ethane.

B.结果以聚甘氨酸为接头的串联TMP二聚体。 B. Results TMP tandem dimer poly-glycine linker. 相互连接的TMP二聚体的设计是基于TMP二聚体形式是与c-Mpl(TPO受体)有效相互作用的需要这一假设,并且根据在受体存在的条件下,它们相互连接的形式,两个TMP分子被设计成在C和N-末端紧密相连,这样就不会影响整个二聚体分子的构型。 TMP dimers was based on the design of interconnected TMP dimer with c-Mpl (TPO receptor) requires the assumption that the effective interaction, and depending on the presence of the receptor under conditions which are interconnected to form , two TMP molecules are designed to be closely linked to the N- and C-terminus, which does not affect the configuration of the entire heterodimeric molecules. 很清楚,串联样连接的二聚体的活性还有赖于适宜的长度和接头成分的选择,该接头将两个TMP单体C和N-末端连接在一起。 Clearly, the activity of the dimer are connected in series like the appropriately selected also depends on the length and composition of the linker, the linker will link two monomer C-terminal and N- TMP. 由于没有TMP与c-Mpl受体结合的结构资料,因此合成了接头含有0-10个和14个甘氨酸残基(表1)的一系列二聚体肽。 Since there is no information on the structure TMP and c-Mpl receptor binding, thus, a series of dimeric peptide linker comprising from 0 to 10 and 14 glycine residues (Table 1). 之所以选择甘氨酸,是由于它的简单性和灵活性。 Glycine was chosen, because of its simplicity and flexibility. 灵活的聚甘氨酸肽链可以允许两个连接在一起的TMP分子自由折叠以形成需要构型,而空间位阻型氨基酸序列则可能形成不需要的二级结构,而其阻滞性则可能干扰二聚体肽与受体的正常结合。 Flexible polyglycine peptide chain may allow two TMP molecules joined together to form the free folded configuration needs, while steric hindrance type amino acid sequence may form unwanted secondary structure, and may interfere with the arrest of two normal-mer peptides binding to the receptor.

可以通过Fmoc或t-Boc化学的常规固相肽合成法(Merrifiled,RB,美国化学协会杂志,85:2149(1963))得到合成的肽。 By conventional solid phase peptide synthesis Fmoc or t-Boc chemistry (Merrifiled, RB, Journal of American Chemical Society, 85: 2149 (1963)) to give a synthetic peptide. 与合成C末端连接的平行二聚体(序列2)不同,该平行二聚体需要应用直角保护的赖氨酸残基作为起始支点来构建两条假对称肽链(Cwirla,SE等,科学,276:1696-1699(1997)),合成我们的串联二聚体是一个从C末端到N末端的简单、逐步完成的连续肽链合成过程。 Is connected in parallel with the synthetic dimer of the C-terminus (SEQ ID 2) different, it needs to be applied at right angles to the parallel dimer protected lysine residue as the initial fulcrum pseudo symmetry to build two peptide chains (Cwirla, SE et al, Science , 276: 1696-1699 (1997)), the synthesis of our tandem dimers was a C-terminus to the N-terminus from a simple, continuous peptide chain synthesis is gradually completed. 由于二聚体化的TMP较C末端二聚体具有非常显著的增殖活性,而非结合亲和力(Cwirla,SE等,科学,276:1696-1699(1997)),合成的肽直接用TPO依赖性细胞增殖法检测生物学活性,该方法使用一种转染了全长c-Mp1(Palacios,R.等,细胞,41:727(1985))的IL-3依赖型鼠32D克隆细胞。 Since dimerization of TMP has a very significant proliferative activity than the C-terminus of the dimer, rather than binding affinity (Cwirla, SE et al., Science, 276: 1696-1699 (1997)), direct peptide synthesis using TPO-dependent detecting a biological activity of proliferation method, which uses a transfected with full-length c-Mp1 (Palacios, R et al., cell, 41: 727 (1985)) of the murine IL-3 dependent 32D cell clones. 如试验结果所示(见下表1),应用这种细胞增殖法,所有聚甘氨酸连接的串联二聚体都较单体显示了1000倍以上的(刺激增殖)潜力,甚至较C末端二聚体也有显著增加。 As shown in the test results (see Table 1), use of such cell proliferation method, all the polyglycine tandem dimer connected shows are more than 1000 times monomer (stimulating proliferation) potential, and even more C-terminal two polyethylene body also significantly increased. 在我们的方法中,C末端二聚体的绝对活性低于天然TPO,这与以前C末端二聚体与天然配体活性一致的报道发现相左(Cwirla,SE等,科学,276:1696-1699(1997))。 In our approach, the C-terminal dimer absolute activity lower than natural TPO, which found that odds (Cwirla, SE et al., Science, 276 with previous C-terminal dimer natural ligand activity consistent reports: 1696-1699 (1997)). 这也许是由于在两种方法中所用条件不同导致的。 This may be due to differences in the conditions used in the two methods result. 但是,在同一试验中,串联二聚体(第一个单体的C末端与第二个单体的N末端相连)与平行二聚体(第一个单体的C末端与第二个单体的C末端相连)活性的差异清楚的显示了串联二聚体产品较平行二聚体产品的优越性。 However, in one test, a tandem dimer (N-terminus and C-terminus of the second monomer is a first monomer linked) parallel dimer (C-terminus of the first monomer and a second single differences in activity C terminus of the connected) clearly shows the superiority of tandem dimer products than parallel dimer products. 我们有兴趣的注意到,接头宽范围的长度变化均可耐受。 We note that there is interest, the change in length of the joint can withstand a wide range. 选定TMP单体的最佳接头(序列1)明显是由8个甘氨酸组成。 Best linker selected TMP monomers (SEQ ID 1) is composed of eight distinct glycine.

其他串联二聚体。 Other tandem dimers. 在这第一系列串联TMP二聚体之后,又设计了几个其他分子,它们或者是接头不同,或者是在单体中含有修饰。 After this first series of TMP tandem dimers, several other molecules and design, which are either different linkers or containing modifications in the monomer. 肽13是这些分子中的第一个,拥有含GPNG的接头,GPNG是一个已知具有形成β-折叠型二级结构的高倾向性的序列。 Peptide 13 is the first of these molecules, with a linker containing GPNG, GPNG is a sequence known to have a high propensity to form a folded β- type secondary structure. 尽管所述肽较单体(刺激增殖)潜力提高大约100倍,但较GGGG-连接类似物的活性低10倍以上。 Although the peptide more monomers (stimulating proliferation) potential to improve about 100 times, but activity than analog connection GGGG- lower than 10 times. 因此,在接头区引入相对僵硬的β-折叠似乎可导致这种短接头形式最佳激动剂构型的轻微破坏。 Thus, the introduction of a relatively rigid β- folded linker region appears to result in optimal agonist configuration slight damage this short linker form.

在随机肽文库中分离的活性肽中,色氨酸9是TMP序列的一个高度保守残基。 Random peptide libraries isolated active peptides, tryptophan 9 is a highly conserved residue of TMP sequence. 在EPO模拟肽同型序列中也存在一个高度保守的色氨酸,并发现这个色氨酸残基参与了两个EPO模拟肽(EMPs)间疏水核心的形成,并与EPO受体的疏水相互作用有关(Livnah,O.等,科学,273:464-471(1996))。 There is also a highly conserved Trp in the same type of EPO mimetic peptide sequences, and found that the two tryptophan residues are involved in the EPO mimetic peptides (EMPs) is formed between the hydrophobic core, and hydrophobic interactions with the EPO receptor about (et Livnah, O, Science, 273: 464-471 (1996)). 通过推理,考虑TMP中的色氨酸9残基可能在肽配体二聚体化过程中具有相似功能,为了调整并评估两个吲哚环之间存在的非共价疏水力效应,构建了几个在色氨酸位点发生了突变的类似物。 By inference, considering 9 TMP tryptophan residues might have a similar function in dimerization of the peptide ligand in the process, in order to adjust and to assess the effect of hydrophobic non-covalent forces exist between the two indole rings, constructed several mutations occurred in tryptophan analogs site. 因此在肽14中,两个TMP单体中的色氨酸均被半胱氨酸取代,并通过氧化作用在两个半胱氨酸残基间形成分子内二硫键,这是为了模仿在肽二聚体化过程中在两个色氨酸间形成的疏水作用。 Therefore, in peptide 14, the two TMP monomers tryptophan cysteine ​​are substituted, to form an intramolecular disulfide bond between the two cysteine ​​residues by oxidation, which is to mimic the hydrophobic interaction process of peptide dimer formed between two tryptophan. 肽15是肽14的还原形式。 Peptide 15 is the reduced form of peptide 14. 在肽16中,两个色氨酸残基被丙氨酸取代。 In peptide 16, the two Trp residues were substituted with alanine. 如试验资料所示,所有这三种类似物都是没有活性的。 As shown in the experimental data, all three analogs were inactive. 这些资料进一步显示,色氨酸不仅对二聚体形成很重要,对保持TPO模拟肽的活性也至关重要。 These data further show that tryptophan is important not only for dimer formation, active in maintaining the TPO mimetic peptide is also crucial.

下面两个肽(肽17a和18)在它们接头的第8个氨基酸上含有赖氨酸或半胱氨酸残基。 The following two peptides (peptide 17a, and 18) containing a lysine or a cysteine ​​residue at the 8 amino acid linker thereof. 这两个化合物是两个聚乙二醇化的肽(肽19和20)的前体,其中,赖氨酸或半胱氨酸侧链被聚乙二醇(PEG)部分修饰。 Both compounds are peptides (peptide 19 and 20) of the two PEGylated precursor, wherein the lysine or cysteine ​​side chains (PEG), polyethylene glycol partially modified. 决定将PEG部分引入一个相对较长的接头的中间部分,这样,这一巨大的PEG组分(5kDa)就可以远离肽分子重要的结合位点。 We decided to introduce a relatively long middle portion of the PEG linker portion, so that a huge component PEG (5kDa) peptide molecules can be remote from the important binding sites. PEG是一种已知的生物兼容性聚合物,作为一种共价修饰物广为应用,可以改善基于肽和蛋白质的药品的药代动力学特征。 PEG is a known biocompatible polymer, as a covalent modifier widely used, can improve the pharmacokinetics of drugs based on peptides and proteins.

为了更方便的使合成或重组肽聚乙二醇化,设计了一种基于液相的模式方法。 In order to facilitate the synthetic or recombinant pegylated peptides, a pattern design method based on the liquid phase. 该方法基于目前已成熟建立的化学选择配基策略,该策略使一对可以相互发生反应的功能基发生特定反应。 The selection method based on chemical ligands has been well-established policy, the policy of the pair of functional groups can react with each other occurrence of a particular reaction. 因此,对肽19来说,先给肽17b的赖氨酸侧链应用溴乙酰基团预激活,以适应与巯基衍生的PEG发生反应。 Thus, peptide 19, the lysine side chain give peptide 17b applications preactivation bromoacetyl group, a mercapto group in order to adapt the derivatized PEG react. 为了达到这一目的,应用一个直角保护基团Dde来保护赖氨酸□-氨基。 For this purpose, at a right angle application Dde protective group protected lysine □ - amino. 一旦整个肽链聚合,就要应用t-Boc对N端氨基进行再保护。 Once the whole peptide chain polymerization, it is necessary to use the N-terminal t-Boc protected amino group again. 然后除去Dde以允许溴乙酰化。 Dde was then removed to allow bromoacetylated. 这一策略可以生产出高质量的原始肽,该肽可通过常规的反向HPLC容易的得到纯化。 This strategy can produce high-quality original peptide, the peptide can be easily purified by conventional reversed HPLC. 肽与巯基修饰的PEG的连接发生在pH8的缓冲液中,反应在30分钟内完成。 Peptide thiol-modified PEG connection occurs in buffer pH8, the reaction was complete within 30 minutes. 应用MALDI-MS分析聚乙二醇化的纯化物质,出现一个特征性钟型光谱,在两个邻近峰之间有一个44Da的增量。 Application of MALDI-MS analysis of purified pegylated species, appears a characteristic bell-shaped spectrum with an increment 44Da between two adjacent peaks. 对PEG-肽20来说,在接头区有一个半胱氨酸残基,其侧链上的巯基可以作为含有马来酰亚胺的PEG的连接位点。 Of PEG- peptide 20, a cysteine ​​residue in a linker region which a mercapto group in the side chain can be used as attachment sites containing PEG maleimide. 可以应用相同的条件聚乙二醇化该肽。 You can apply the same conditions as the pegylated peptides. 如试验资料显示,这两种聚乙二醇化的肽,体外生物活性甚至较它们未聚乙二醇化的肽高。 The experimental data show that these two pegylated peptides, in vitro biological activity even higher than their unpegylated peptide.

肽21在其具有8个氨基酸的接头上存在一个潜在糖基化功能区,NGS。 Peptide 21 there is a potential glycosylation ribbon on which an 8 amino acid linker having, NGS. 由于示例串联二聚体是由天然氨基酸通过肽键连接组成的,因此在真核细胞系统中表达这样一个分子,可以产生在天门冬酰胺羧胺侧链上添加有碳水化合物部分的糖蛋白。 Since the exemplary tandem dimers are connected to form a peptide bond, so that expression of such a molecule in a eukaryotic cell system, can be added to the produced carboxamide asparagine side chain carbohydrate portion of the glycoprotein from a natural amino acid through. 糖基化是一个常见的翻译后修饰方法,可以通过增加蛋白质的水溶性和体内稳定性,对一个既定蛋白质的生物活性产生很多积极影响。 Glycosylation is a common post-translational modification methods can produce many positive effects on the biological activity of a given protein by increasing the water solubility and stability of the protein in vivo. 如试验资料所示,将糖基化功能区整合进入接头,依然可以维持高生物活性。 As shown in the experimental data, glycosylation ribbon incorporated into linker, can still maintain high biological activity. 合成的潜在糖蛋白前体与-(甘氨酸)8-连接的类似物具有相当的活性。 Potential synthetic glycoprotein precursor and - (Gly) 8- linked analog having a comparable activity. 一旦糖基化,这种肽将与聚乙二醇化的肽具有相同的活性,因为PEG和碳水化合物部分显示了相似的理化特性。 Once glycosylated, this peptide will have the same activity as the pegylated peptides, because PEG and a carbohydrate moiety showed similar physical and chemical properties.

最后一种肽是二聚体的二聚体。 The last peptide is a dimer of dimers. 它通过氧化肽18获得,这一方法可以使位于接头上的两个半胱氨酸残基形成分子内二硫键。 It is obtained by oxidizing peptide 18, this approach allows two cysteine ​​residues located on the linker intramolecular disulfide bond formation. 这种肽是为了表明TMP四聚体也具有活性的可能性而设计的。 This is to show that TMP peptide tetramers having also the possibility of active designed. 试验资料显示这种肽在调整摩尔的基础上,并不比平均串联二聚体更具活性,这一结果间接支持了TMP的活性形式其实是二聚体的想法,否则将串联二聚体再二聚体化会对生物活性产生进一步影响。 Test data show that this peptide adjustment molar basis, is not more active than an average tandem dimer, the results indirectly support the active form of TMP is actually the idea of ​​a dimer, otherwise tandem dimer dichotomized trimerization have a further impact biological activity.

下表1总结了应用上述体外试验方法得出的上述化合物的相对活性(相对潜力)。 Table 1 below summarizes the relative activity of the above compounds obtained using the above in vitro test methods (relative potential).

表1化合物相对(增殖刺激)潜力(EC50)TPO 4.0TMP单体(序列1) 1.0TMP CC二聚体(序列2) 3.5TMP-(Gly)n-TMP:1 n=0 4.52 n=1 4.03 n=2 4.04 n=3 4.05 n=4 4.06 n=5 4.07 n=6 4.08 n=7 4.09 n=8 4.510 n=9 - 4.011 n=10 4.012 n=14 4.013 TMP-GPNG-TMP(序列10) 3.0 Table 1 Compound relative (to cell proliferation) Potential (EC50) TPO 4.0TMP monomer (SEQ ID 1) 1.0TMP CC dimer (SEQ ID 2) 3.5TMP- (Gly) n-TMP: 1 n = 0 4.52 n = 1 4.03 n = 2 4.04 n = 3 4.05 n = 4 4.06 n = 5 4.07 n = 6 4.08 n = 7 4.09 n = 8 4.510 n = 9 - 4.011 n = 10 4.012 n = 14 4.013 TMP-GPNG-TMP (sequence 10) 3.0 (序列11)15IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA 0.5(序列12)16IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA 0.5(序列13)17a TMP-GGGKGGGG-TMP (序列14) 4.017b TMP-GGGK(BrAc)GGGG-TMP (序列15) ND18TMP-GGGCGGGG-TMP (序列16) 4.019TMP-GGGK(PEG)GGGG-TMP (序列17) 5.020TMP-GGGC(PEG)GGGG-TMP (序列18) 5.021TMP-GGGNGSGG-TMP (序列19) 4.022TMP-GGGCGGGG-TMP (序列20) 4.0|TMP-GGGCGGGG-TMP备注:表1中的数字大约表示活性的对数,因此活性“1”和“4”之间的差异大约是1000倍。 (SEQ ID 11) 15IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA 0.5 (sequence 12) 16IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA 0.5 (sequence 13) 17a TMP-GGGKGGGG-TMP (sequence 14) 4.017b TMP-GGGK (BrAc) GGGG-TMP (sequence 15) ND18TMP -GGGCGGGG-TMP (sequence 16) 4.019TMP-GGGK (PEG) GGGG-TMP (sequence 17) 5.020TMP-GGGC (PEG) GGGG-TMP (sequence 18) 5.021TMP-GGGNGSGG-TMP (sequence 19) 4.022TMP-GGGCGGGG -TMP (sequence 20) 4.0 | TMP-GGGCGGGG-TMP NOTE: in table 1, numerals indicate approximately the logarithm of the activity, and therefore the difference in activity between "1" and "4" is approximately 1000-fold. 0.5的增量是中间点,因此活性“1”和“3.5”之间的差异大约是500倍。 Increment of 0.5 is an intermediate point, so the difference between the active "1" and "3.5" is approximately 500-fold. “ND”的意思是没有检测。 "ND" means not detected.

II.下面将介绍这里公开的一些第二组化合物的制备方法示例。 II. The following example describes the preparation of some of the second group of compounds disclosed herein.

A.如图6C所示Fc融合化合物的制备编码人IgG1Fc段的DNA序列与TPO模拟肽二聚体(序列34)框内融合,置于如下所示表达载体质粒pAMG21中的luxPR启动子控制下。 The DNA sequence and the TPO mimetic peptide dimer (SEQ ID 34) was prepared encoding human IgG1Fc fusion frame segment shown in FIG. 6C A. FIG Fc fusion compound luxPR promoter placed under the control plasmid expression vector pAMG21 as follows in .

融合基因通过标准PCR技术构建。 Construction of the fusion gene by standard PCR techniques. PCR反应的模板是含有Fc序列和编码序列34化合物剩余物的合成基因的融合载体。 Templates for PCR reactions were the fusion vector containing the Fc sequence of the synthetic gene and the coding sequence of 34 residue compounds. 该合成基因通过下面显示的4个重叠寡核苷酸构建:1830-52 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCGACT CTG CGT CAG TGG CTG GCT GCT CGT GCT(序列35)1830-53 ACC TCC ACC ACC AGC ACG AGC AGC CAGCCA CTG ACG CAG AGT CGG ACC (序列36)1830-54 GGT GGT GGA GGT GGC GGC GGA GGT ATT GAG GGCCCA ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA(序列37)1830-55 AAA AAA AGG ATC CTC GAG ATT ATG CGC GTG CTGCAA GCC ATT GGC GAA GGG TTG GGC CCT CAA TACCTC CGC CGC C (序列38)这4个寡核苷酸经过退火形成下面所示的双链结构。 The synthetic gene was shown by the following four overlapping oligonucleotides Construction: 1830-52 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCGACT CTG CGT CAG TGG CTG GCT GCT CGT GCT (sequence 35) 1830-53 ACC TCC ACC ACC AGC ACG AGC AGC CAGCCA CTG ACG CAG AGT CGG ACC (sequence 36) 1830-54 GGT GGT GGA GGT GGC GGC GGA GGT ATT GAG GGCCCA ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA (sequence 37) 1830-55 AAA AAA AGG ATC CTC GAG ATT ATG CGC GTG CTGCAA GCC ATT GGC GAA GGG TTG GGC CCT CAA TACCTC CGC CGC C (sequence 38) forming the four shown in the following oligonucleotides annealed double-stranded structure.

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT1--------+---------+---------+---------+---------+---------+60CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGAK G G G G G I E G P T L R Q W L A A R AGGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA---------+---------+---------+---------+---------+---------+120CCACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTG G G G G G G G I E G P T L R Q W L A ACGCGCA---------------------------148GCGCGTATTAGAGCTCCTAGGAAAAAAAR A *序列39[线性对称寡核苷酸1830-52和1830-54]序列40[线性对称寡核苷酸1830-53和1830-55]以及序列41[编码氨基酸序列]这一双链结构应用PCR反应扩增,以1830-52和1830-55作为正义和反义引物。 AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT1 -------- + --------- + --------- + --------- + --------- + --------- + 60CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGAK GGGGGIEGPTLRQWLAAR AGGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA --------- + --------- + --------- + ------- - + --------- + --------- + 120CCACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTG GGGGGGGIEGPTLRQWLA ACGCGCA ------------------------ --- 148GCGCGTATTAGAGCTCCTAGGAAAAAAAR A * sequence 39 [symmetric linear oligonucleotides 1830-52 and 1830-54] sequence 40 [symmetric linear oligonucleotides 1830-53 and 1830-55] and [encoded amino acid sequence] this sequence of 41 bis chain was amplified by PCR to 1830-52 and 1830-55 as the sense and antisense primers.

分子的Fc部分通过PCR反应应用引物扩增Fc DNA产生。 Generating Fc Fc portion of the DNA molecule by PCR amplification of the primers.

1216-52 AAC ATAAGT ACC TGTAGG ATCG_ (序列42)1830-51 TTCGATACCACCACCTCCACCTTTACCCGGAG-ACAGGGAGAGGCTCTTCTGC (序列43)寡核苷酸1830-51和1830-52含有重叠的24个核苷酸,允许通过应用另外的引物1216-52和1830-55,在第三个反应中将上述PCR产物结合,从而将两个基因以正确的阅读框融合在一起。 1216-52 AAC ATAAGT ACC TGTAGG ATCG_ (sequence 42) 1830-51 TTCGATACCACCACCTCCACCTTTACCCGGAG-ACAGGGAGAGGCTCTTCTGC (sequence 43) oligonucleotides 1830-51 and 1830-52 contain overlapping 24 nucleotides, allows application of additional primers 1216- 1830-55 and 52, the above product was combined in a third PCR reaction, so that the two genes together in the correct reading frame.

最终的PCR基因产物(全长融合基因)用限制性内切酶XbaI和BamHI消化,然后与同样经过XbaI和BamHI消化的载体Pamg21(见下)连接。 The final PCR gene product (the full length fusion gene) was cut with the enzymes XbaI and BamHI restriction, and then connected to the carrier through the same Pamg21 digested with XbaI and BamHI (see below). 用连接DNA转化宿主细胞大肠杆菌2596株(GM221,见下述)。 Connected to a host cell transformed with the DNA of Escherichia coli 2596 (GM221, see below). 筛选具有产生重组蛋白产品能力并拥有正确核苷酸序列的融合基因的克隆。 Screening of clones having the ability to produce the recombinant protein product and to possess the gene fusion in the correct nucleotide sequence. 蛋白表达水平应用50ml摇瓶研究检测。 Protein expression detected application 50ml shake flask studies. 全菌体溶菌液用考马斯染色的PAGE凝胶分析融合基因的表达。 Whole cell lysates of the fusion gene expression analysis using Coomassie stained PAGE gel.

融合蛋白的氨基酸序列及相应的核苷酸序列如下所示:XbaI|TCTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGACAAAACTCACACATGTC1 ---------+---------+---------+---------+---------+---------+ 60AGATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCTGTTTTGAGTGTGTACAGM D K T H T C PCACCTTGTCCAGCTCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAAC61 ---------+---------+---------+---------+---------+---------+ 120GTGGAACAGGTCGAGGCCTTGAGGACCCCCCTGGCAGTCAGAAGGAGAAGGGGGGTTTTGP C P A P E L L G G P S V F L F P P K PCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA121 ---------+---------+---------+---------+---------+---------+ 180GGTTCCTGTGGGAGTACTAGAGGGCCTGGGGACTCCAGTGTACGCACCACCACCTGCACTK D T L M I S R T P E V T C V V V D V SGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG181 ---------+---------+---------+---------+---------+---------+ 240CGGTGCTTCTGGGACTCCAGTTCAAGTTGACCATGCACCTGCCGCACCTCCACGTATTACH E D P E V K F N W Y V Fusion protein amino acid sequence and corresponding nucleotide sequences are shown below: XbaI | TCTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGACAAAACTCACACATGTC1 --------- + --------- + --------- + - -------- + --------- + --------- + 60AGATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCTGTTTTGAGTGTGTACAGM DKTHTC PCACCTTGTCCAGCTCCGGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAAC61 --------- + -------- - + --------- + --------- + --------- + --------- + 120GTGGAACAGGTCGAGGCCTTGAGGACCCCCCTGGCAGTCAGAAGGAGAAGGGGGGTTTTGP CPAPELLGGPSVFLFPPK PCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA121 ----- ---- + --------- + --------- + --------- + --------- + ----- ---- + 180GGTTCCTGTGGGAGTACTAGAGGGCCTGGGGACTCCAGTGTACGCACCACCACCTGCACTK DTLMISRTPEVTCVVVDV SGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG181 --------- + --------- + --------- + --------- + - ------- + --------- + 240CGGTGCTTCTGGGACTCCAGTTCAAGTTGACCATGCACCTGCCGCACCTCCACGTATTACH EDPEVKFNWYV D G V E V H N ACCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA241 ---------+---------+---------+---------+---------+---------+ 300GGTTCTGTTTCGGCGCCCTCCTCGTCATGTTGTCGTGCATGGCACACCAGTCGCAGGAGTK T K P R E E Q Y N S T Y R V V S V L TCCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAGGTCTCCAACAAAAG301 ---------+---------+---------+---------+---------+---------+ 360GGCAGGACGTGGTCCTGACCGACTTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCV L H Q D W L N G K E Y K C K V S N K A DGVEVHN ACCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA241 --------- + --------- + --------- + --------- + -------- - + --------- + 300GGTTCTGTTTCGGCGCCCTCCTCGTCATGTTGTCGTGCATGGCACACCAGTCGCAGGAGTK TKPREEQYNSTYRVVSVL TCCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAGGTCTCCAACAAAAG301 --------- + --------- + --------- + ----- ---- + --------- + --------- + 360GGCAGGACGTGGTCCTGACCGACTTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCV LHQDWLNGKEYKCKVSNKA

CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC361 ---------+---------+---------+---------+---------+---------+ 420GGGAGGGTCGGGGGTAGCTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGL P A P I E K T I S K A K G Q P R E P QAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCT421 ---------+---------+---------+---------+---------+---------+ 480TCCACATGTGGGACGGGGGTAGGGCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGAV Y T L P P S R D E L T K N Q V S L T CGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC481 ---------+---------+---------+---------+---------+---------+ 540CGGACCAGTTTCCGAAGATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGL V K G F Y P S D I A V E W E S N G Q PCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT541 ---------+---------+---------+---------+---------+---------+ 600GCCTCTTGTTGATGTTCTGGTGCGGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGAE N N Y K T T P P V L D S D G S F F L YACAGCAAGCTCACCGT CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC361 --------- + --------- + --------- + --------- + --------- + --------- + 420GGGAGGGTCGGGGGTAGCTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGL PAPIEKTISKAKGQPREP QAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCT421 --------- + --------- + --------- + ------ --- + --------- + --------- + 480TCCACATGTGGGACGGGGGTAGGGCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGAV YTLPPSRDELTKNQVSLT CGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC481 --------- + --------- + --- ------ + --------- + --------- + --------- + 540CGGACCAGTTTCCGAAGATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGL VKGFYPSDIAVEWESNGQ PCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT541 --------- + --------- + --------- + --------- + --------- + --------- + 600GCCTCTTGTTGATGTTCTGGTGCGGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGAE NNYKTTPPVLDSDGSFFL YACAGCAAGCTCACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG601 ---------+---------+---------+---------+---------+---------+ 660TGTCGTTCGAGTGGCACCTGTTCTCGTCCACCGTCGTCCCCTTGCAGAAAGAGTACGAGGCS K L T V D K S R W Q Q G N V F S C S VTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA661 ---------+---------+---------+---------+---------+---------+ 720ACTACGTACTCCGAGACGTGTTGGTGATGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATM H E A L H N H Y T Q K S L S L S P G KAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTG721 ---------+---------+---------+---------+---------+---------+ 780TTCCACCTCCACCACCATAGCTTCCAGGCTGAGACGCGTCACCGACCGACGAAGCACGACG G G G G I E G P T L R Q W L A A R A GGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCAC781 ---------+---------+---------+---------+---------+---------+ 840CACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTGG G G G G G G I E G P T L R Q W L A A RBamHI|GCGCATAATCTCGAGGATCCG841 GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG601 --------- + --------- + --------- + --------- + --------- + --------- + 660TGTCGTTCGAGTGGCACCTGTTCTCGTCCACCGTCGTCCCCTTGCAGAAAGAGTACGAGGCS KLTVDKSRWQQGNVFSCS VTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA661 --------- + --------- + --------- + ------ --- + --------- + --------- + 720ACTACGTACTCCGAGACGTGTTGGTGATGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATM HEALHNHYTQKSLSLSPG KAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTG721 --------- + --------- + --- ------ + --------- + --------- + --------- + 780TTCCACCTCCACCACCATAGCTTCCAGGCTGAGACGCGTCACCGACCGACGAAGCACGACG GGGGIEGPTLRQWLAARA GGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCAC781 --------- + --------- + --------- + --------- + --------- + --------- + 840CACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTGG GGGGGGIEGPTLRQWLAA RBamHI | GCGCATAATCTCGAGGATCCG841 ---------+---------+- 861CGCGTATTAGAGCTCCTAGGCA序列44[上示单链,阅读方向5'→3']序列45[上示单链,阅读方向3'→5']以及序列46[编码的氨基酸序列]pAMG21表达质粒pAMG21可通过ATCC获得,其保藏号为98113,保藏日期为1996年7月24日。 --------- + --------- + - 861CGCGTATTAGAGCTCCTAGGCA 44 sequence [shown on a single strand, the reading direction of 5 '→ 3'] Sequence 45 [shown on a single strand, reading direction 3 ' → 5 '] and a sequence 46 [the encoded amino acid sequence] pAMG21 the expression plasmid pAMG21 can be obtained by ATCC, accession number 98113, with a deposit date July 24, 1996.

GM221(Amgen宿主株#2596)Amgen宿主株#2596是一种业已经过修饰的大肠杆菌K-12株,含有位于早ebg区的温度敏感λ抑制子cI 857s7和位于晚ebg区的lacIQ抑制子(68分钟)。 GM221 (Amgen host strain # 2596) Amgen host strain # 2596 is an already modified coli K-12 strain containing a temperature sensitive λ early ebg region located repressor cI 857s7 located late ebg region lacIQ repressor ( 68 minutes). 这两个抑制子基因的存在使这种宿主可以广泛应用于表达系统,但这两种抑制子与luxPR的表达无关。 Presence of these two repressor genes allows the expression of such host systems can be widely used, but both promoter and inhibiting expression regardless of the luxPR. 未转化的宿主无抗生素耐药性。 Untransformed host no antibiotic resistance.

cI857s7基因的核糖体结合位点经过修饰,包括一个增强的RBS。 CI857s7 gene ribosomal binding site has been modified, including an enhanced RBS. 它被插入ebg操纵子中,该操纵子位于核苷酸位点1170和1411之间,其计数与去除了插入ebg序列的基因文库编号为M64441Gb_Ba的一致。 It is inserted into the ebg operon, which operon is located in which the count coincidence between nucleotide positions 1170 and 1411 in addition to the insertion ebg sequence number M64441Gb_Ba the gene library.

通过被称之为MMebg-cI857s7增强RBS#4的重组噬菌体将构建体导入F'tet/393染色体中。 Enhanced RBS # 4 by the recombinant phage called MMebg-cI857s7 the construct into F'tet / 393 chromosome. 经重组和分离后,只有上述染色体插入遗留在菌体中。 After recombination and separated, and only the cells are left in the chromosome is inserted. 将之重新命名为F'tet/GM101。 It will be renamed F'tet / GM101.

然后,F'tet/GM101通过导入lacIQ构建体进行修饰,该构建体被插入ebg操纵子中,该操纵子位于核苷酸位点2493和2937之间,其计数与去除了插入ebg序列的基因文库编号为M64441Gb_Ba的一致。 Then, F'tet / GM101 be modified by introducing lacIQ construct, the construct is inserted into the ebg operon, which operon is located between nucleotide positions 2493 and 2937, which count removes ebg sequence inserted gene library number M64441Gb_Ba consistent.

通过被称之为AGebg-LacIQ#5的重组噬菌体将构建体导入F'tet/GM101染色体中。 By being called AGebg-LacIQ # 5 recombinant phage in the construct introduced into F'tet / GM101 chromosome. 经重组和分离后,只有上述染色体插入遗留在菌体中。 After recombination and separated, and only the cells are left in the chromosome is inserted. 将之重新命名为F'tet/GM221。 It will be renamed F'tet / GM221. 然后用溶于LB浓度为25μg/ml的吖啶橙将F'tet附加体从菌株中去除。 LB and then dissolved at a concentration of 25μg / ml of acridine orange is removed from the strain F'tet episome. 去除F'tet附加体的菌株经鉴定为四环素敏感,并以GM221命名保藏。 Removing the identified strain F'tet episome tetracycline sensitive, and is named GM221 deposited.

质粒pAMG21含有Fc融合构建体(在这里称之为pAGM21-Fc-TMP-TMP),含有该质粒的宿主株GM221保藏于ATCC,保藏号为98957,保藏时间为1998年10月22日。 The plasmid containing the Fc fusion constructs pAMG21 (referred to herein as pAGM21-Fc-TMP-TMP), the host strain GM221 containing plasmid deposited with ATCC, accession number 98957, deposited time is October 22, 1998.

表达。 expression. 在含有50μg/ml卡那霉素的Luria Broth培养基中培养的含pAGM21-Fc-TMP-TMP的大肠杆菌GM221,诱导前37℃孵育。 Cultured in Luria Broth medium containing 50μg / ml kanamycin in E. coli GM221 containing pAGM21-Fc-TMP-TMP, and incubated at 37 ℃ before induction. 在向培养基中添加合成自动诱导剂N-(3-氧己酰)-DL-同型丝氨酸内酯至终浓度为20ng/ml后,在luxPR启动子的作用下,诱导Fc-TMP-TMP基因产物表达开始,培养基在37℃进一步孵育3小时。 After the addition of the synthetic inducer automatically N- (3- oxo hexanoyl) -DL-homoserine lactone to a final concentration of 20ng / ml to the medium, under the action of luxPR promoter induced Fc-TMP-TMP gene product expression begins, the medium further incubated at 37 ℃ 3 hours. 3小时后,显微镜检查细菌培养基中内含体的存在,并离心收集。 After 3 hours, the bacterial culture microscopic examination the presence of inclusion bodies and collected by centrifugation. 在诱导培养基中观察到有折射能力的内含体表示,在大肠杆菌中产生的Fc-TMP-TMP最有可能是不溶性的片段。 Observed induction medium with a refractive power in inclusion bodies expressed, Fc-TMP-TMP produced in E. coli is most likely to be insoluble fragments. 将菌体颗粒再悬浮于含10%β-巯基乙醇的Laemmli样品缓冲液中直接溶解,然后通过SDS-PAGE分析。 The cell pellet was resuspended in Laemmli sample buffer containing 10% β- mercaptoethanol directly dissolved and then analyzed by SDS-PAGE. 在SDS-PAGE凝胶上,可以观察到大约30kDa的考马斯加强染色带。 On SDS-PAGE gels, it was observed Coomassie stained band of approximately 30kDa reinforcement. 预期的基因产物长度为269个氨基酸,预期分子量大约为29.5kDa。 Gene product of the expected length of 269 amino acids, is expected molecular weight of approximately 29.5kDa. 在标准10L规模的批量条件下进行发酵,获得的Fc-TMP-TMP的表达水平与在小规模下相似。 Fermented under standard conditions 10L batch size, the expression levels of Fc-TMP-TMP obtained in a similar small scale.

Fc-TMP-TMP的纯化通过高压匀浆(14000PSI通过2次)将菌体在水中(1/10)碎裂,并通过离心(在J-6B离心机中以4200转/分离心1小时)收获内含体。 Fc-TMP-TMP was purified by high pressure homogenization (14000PSI by 2 times) the cells in water (1/10) fragmentation by centrifugation (in J-6B centrifuge at 4200 rpm / centrifuged 1 hour) harvest inclusion bodies. 以1/10的比例将内含体溶解于pH8.7的6M胍,50mM Tris,8mM DDT中1小时。 The contents in a ratio of 1/10 of pH8.7 was dissolved in 6M guanidine, 50mM Tris, 8mM DDT for 1 hour. 溶解混合物用pH8.5的2M尿素,50mM Tris,160mM精氨酸,3mM半胱氨酸稀释20倍。 The mixture was dissolved in pH8.5 with 2M urea, 50mM Tris, 160mM arginine, 3mM cysteine ​​was diluted 20-fold. 混合物在冷环境下搅拌过夜。 The mixture was stirred overnight in the cold environment. 在整个过程的这一点,Fc-TMP-TMP单体亚单位二聚体化,形成具有如图6C所示结构的二硫键连接的化合物。 In this regard, Fc-TMP-TMP monomer subunits dimerize the whole process, to form a compound having a disulfide-linked structure as shown FIG. 6C. 然后通过超滤作用浓缩大约10倍。 Then concentrated approximately 10-fold by ultrafiltration. 之后,再用pH 9的10mM Tris,1.5M尿素稀释3倍。 Thereafter, in 10mM Tris pH 9 and then, 1.5M urea 3-fold dilutions. 然后用乙酸调节混合物pH至5。 The mixture was then adjusted with acetic acid to pH 5. 离心去除沉淀物,将上清装载到用pH 5的20mM NaAc,100mMNaCl平衡的SP-Sepharose快流柱上(蛋白负载浓度为10mg/ml,室温)。 The precipitate was removed by centrifugation, the supernatant was loaded onto a 20mM NaAc pH 5 to, 100 mM NaCl equilibrated SP-Sepharose fast flow column (protein load concentration of 10mg / ml, room temperature). 用溶解于相同缓冲液的100mM-500mM的NaCl以20倍柱容积梯度洗脱蛋白质。 Used dissolved in the same buffer of 100mM-500mM NaCl in 20 column volumes gradient protein. 洗脱液稀释3倍后,装载到pH5的20mM NaAc,150mM NaCl平衡的SP-Sepharose HP柱上(蛋白负载浓度为10mg/ml,室温)。 After the eluate was diluted 3 fold and loaded onto a 20mM NaAc pH5, 150mM NaCl equilibrated SP-Sepharose HP column (protein load concentration of 10mg / ml, room temperature). 用溶解于相同缓冲液的150mM-400mM的NaCl以20倍柱容积梯度洗脱蛋白质。 With NaCl dissolved in the same buffer at 150mM-400mM gradient 20 column volumes of protein. 收集过滤高峰洗脱液。 Collected by filtration peak eluent.

III.下面总结了多种本发明化合物在小鼠体内试验的资料。 III. The following summarizes the information on compounds of the invention in vivo tests in mice.

小鼠。 Mouse. 大约10-12周龄的正常雌性BDF1小鼠。 About 10-12 week old normal female BDF1 mice.

放血方案。 Phlebotomy program. 每组的其中10只小鼠在第0天接受处理,2组在4天后开始,每组总共20只小鼠。 Ten mice per group treated on day 0 accepted, two groups started 4 days, a total of 20 mice per group. 5只小鼠在每一时间点放血,每周至少放血3次。 Five mice bled at each time point, were bled at least 3 times a week. 小鼠用异氟烷麻醉,并通过针刺眼窦放血,放血总量为140-160μl。 Mice were anesthetized with isoflurane and bled through the sinus needle eye, the total amount of blood is 140-160μl. 鼠血在运行鼠血软件的Technicon H1E全血分析仪上计数。 Rat blood count on Technicon H1E blood software running murine whole blood analyzer. 检测的参数有白细胞,红细胞,红细胞压积,血红蛋白,血小板和中性粒细胞。 Detected parameters leukocytes, red blood cells, hematocrit, hemoglobin, platelets and neutrophils.

治疗。 treatment. 小鼠或者接受皮下弹丸注射治疗,或者通过埋植的微渗透泵接受连续7天给药治疗。 Mice received subcutaneous bolus injection or treatment, or accept 7 consecutive days is treated by the implanted micro osmotic pump. 皮下注射给药量为0.2ml。 The amount of administration is subcutaneous injection 0.2ml. 渗透泵插入位于麻醉小鼠肩胛骨之间皮肤上的皮下切口。 Osmotic pump is inserted under the skin incision in the skin between the scapulae of anesthetized mice. 化合物用含0.1%BSA的PBS稀释。 Compound diluted in PBS containing 0.1% BSA. 所有的试验都包括一个对照组,标记为“载体”,只用稀释液处理。 All tests including one control group, labeled "carrier", treated with diluent only. 泵中试验样品的浓度经过调整,因此泵中给药的校正流速可以通过图形显示。 The concentration of the test sample through the pump adjusted so that the flow rate of the pump administration may be corrected by the graphic display.

化合物。 Compound. 一个剂量滴定的化合物通过微渗透泵在7天内给予小鼠。 A dose titration of the compound was administered to mice in 7 days by osmotic micro pumps. 多种化合物以单剂量为100μg/kg的量通过微渗透泵在7天内给予小鼠。 More compounds in a single dose of 100μg / kg and administered to mice within 7 days by osmotic micro pumps. 一些相同的化合物以单次弹丸注射的方式给予小鼠。 Some of the same compound in a single bolus injection is administered to mice manner.

活性试验结果。 Activity test results. 活性试验的结果在图4和5中显示。 The results of activity tests shown in FIGS. 4 and 5. 在应用7天微渗透泵给药的剂量反应试验中(试验数据未给出),序列18化合物的最大效果出现在100μg/kg/天;10μg/kg/天的剂量大约是最大活性的50%,1μg/kg/天是最低剂量,在该试验受体中仍可观察到活性。 Application of mini-osmotic pumps 7 days of administration of dose response experiments (experimental data not shown), the sequence of the maximum effect of 18 compounds appeared in 100μg / kg / day; dose of 10μg / kg / day to about 50% of the maximum activity , 1μg / kg / day was the lowest dose, still observed activity in this assay receptor. 在同一试验中,该化合物10μg/kg/天的剂量大约与100μg/kg/天未聚乙二醇化的rHu-MGDF活性相当。 In the same trial, 10μg / kg / day dose of the compound equivalent to about 100μg / kg / day unpegylated rHu-MGDF in activity.

IV.讨论MGDF与人生长激素(hGH)以相似的方式发挥作用已广为接受,即一分子蛋白配体与两分子受体结合以激活受体(Wells,JA等,生物化学评论纪事,65:609-634(1996))。 IV. MGDF discussions with human growth hormone (hGH) in a similar manner to play a role has been widely accepted that the protein molecule ligand binding to receptor molecules to activate two receptors (Wells, JA et al., Biochemistry comment Chronicle, 65 : 609-634 (1996)). 比之甚小的TMP肽也通过模仿这种相互作用方式发挥作用。 A very small ratio of TMP peptides also play a role in this interaction by imitating the way. 但是本研究显示,这种模仿需要两个TMP分子共同作用,即TMP以CC平行或CN依次共价二聚体的方式,可以较原始单体体外生物活性潜能增加103以上。 However, the present study shows that this requires two interacting mimic TMP molecules, i.e. TMP by CC parallel or sequentially CN covalent dimer way, in vitro biological activity than the original monomers potential increases above 103. 单体相对低下的生物潜能可能是由于没有有效的形成非共价二聚体。 Biotic potential relatively low monomer may be formed no effective non-covalent dimers. 预先形成的共价二聚体可以消除形成非共价二聚体的熵阻障,该非共价二聚体是在两个14残基肽小分子间形成的唯一较弱的非共价相互作用。 Preformed covalent dimer may eliminate the entropy barrier forming a non-covalent dimer, which is the only non-covalent dimer weaker noncovalent each formed between two 14-residue peptide small molecules effect.

我们饶有兴趣的注意到,绝大部分串联二聚体较C末端平行二聚体更具(生物)潜能。 Our interesting to note that most of the tandem dimer more than the C-terminal parallel dimers (biological) potential. 串联二聚体化似乎较CC平行二聚体化赋予分子更好的适应构型。 CC series dimerization seems to be more parallel dimeric molecule confers better adapt to configuration. 串联二聚体看起来不对称的特征可能使它更接近天然配体,作为一种非对称分子,天然配体可能用两个不同的位点与两个相同的受体分子结合。 Tandem dimer appears asymmetrical features may bring it closer to the natural ligand, as an asymmetric molecule, natural ligands may use two different sites with two identical receptor molecules.

引入PEG部分应该得到正视,因为它通过保护蛋白分子避免蛋白水解,延缓通过肾滤过的清除,而增强了修饰肽的体内活性。 PEG should be introduced into the front part, because it avoids the protection protein molecules by proteolysis, delaying clearance by renal filtration, and enhance the in vivo activity of the modified peptide. 在基于细胞的增殖试验中,聚乙二醇化可以进一步增加串联二聚体TMP肽的体外生物活性是一个意外收获。 In the cell-based proliferation assay, PEGylation can be further increased in vitro biological activity of TMP tandem dimer peptide is a bonus.

V.下面总结了多种本发明化合物在猴体内试验的资料。 V. The following summarizes the information on compounds of the invention tested in monkeys.

为了评价皮下给予TMP对雌性恒河猴血液学参数的影响,设计并实施了以下方案。 In order to evaluate the effects on subcutaneous administration of TMP hematological parameters in female rhesus monkeys, the design and implementation of the following embodiment. 试验共设5组,每组3只恒河猴。 A total of 5 test groups of 3 rhesus monkeys. 组1为对照组,给予不含TMP或聚乙二醇化的重组人MGDF(PEG-rHuMGDF)的醋酸盐缓冲液(20mM醋酸钠,0.25mM氯化钠,pH5);组2以下述间隔给予一剂或多剂TMP;组3以下述间隔给予1000μg/kg的TMP;组4以下述间隔给予5000μg/kg的TMP;组5以下述间隔给予100μg/kg的PEG-rHuMGDF。 Group 1 was the control group, administered free of TMP or pegylated recombinant human MGDF (PEG-rHuMGDF) in acetate buffer (20mM sodium acetate, 0.25mM sodium chloride, pH 5); group 2 at intervals following administration one or more doses of the TMP; group 3 in the following intervals administered 1000μg / kg of the TMP; group 4 administered at the following intervals 5000μg / kg of the TMP; 5 group represented by the following intervals administered 100μg / kg of PEG-rHuMGDF.

首次单剂量给药时间被定义为第1周期第0天。 The first single dose was defined as a first time period day 0. 在第2周期中,给药时间为第21,23,25,28,30和32天。 In the second period, for the first time of administration 21,23,25,28,30 and 32 days. 在第3周期,第84天单剂量给药一次,在第4周期,第123天单剂量给药一次。 In the third period, a single dose of 84 days, in the fourth period, a single dose of 123 days. 观察试验动物临床体征,在适应环境期每天1次,在给药日每天3次(给药前,给药30分钟后,给药2-3小时后),在非给药日,每天1次。 Test animals were observed for clinical signs, in an acclimatization period per day, at 3 times daily (prior to dosing, 30 minutes after dosing, 2-3 h after administration) administration, at the non-administration days, 1 day . 从试验开始前7天到恢复期结束,通过给予每个试验动物的食物片数和剩余食物片数计算每天食量。 7 days from the end of the recovery period prior to the test, calculated by the number of food pieces given and the number of remaining food pieces daily food intake of each test animal. 每个试验动物在开始给药方案前测量体重两次,给药期间和恢复期再测量两次。 Each test animal body weight was measured twice before the start of the dosing regimen, administration period and the recovery was measured twice again. 在开始给药前和试验第1,3,5,7,9,11,13,15,20,22,24,26,29,31,33,35,37,39,41,43,45,47,49,55,62,69,76,83,85,87,89,91,93,95,97,99,101,103,105,111,122,124,126,128,130,132,134,136,138,140,142,144,150天分别采集血样1次备用。 Before the start of dosing and Test 1,3,5,7,9,11,13,15,20,22,24,26,29,31,33,35,37,39,41,43,45, 47,49,55,62,69,76,83,85,87,89,91,93,95,97,99,101,103,105,111,122,124,126,128,130,132, blood samples were collected 1 day 134,136,138,140,142,144,150 standby times. 药代动力学分析。 Kinetic analysis of pharmacokinetics. 在给药前和给药后1,4和24小时分别采集0.5ml血清样本。 Prior to dosing and at 1, 4 and 24 hours 0.5ml serum samples were collected. 样本在第0,21,32,84和123天采集,并储存于大约-70℃,直到分析。 In the samples collected on days 0,21,32,84 and 123, and stored at about -70 deg.] C, until analysis. 为分析抗体,在单剂给药前1周和第0(给药前),6,13,20,27,34,41,48,55,62,69,76,83,90,97,104,111,118,129,136,143和150天分别采集血样2ml。 To analyze the antibody prior to administration of a single dose and one week 0 (pre-dose), 6,13,20,27,34,41,48,55,62,69,76,83,90,97,104 , 111,118,129,136,143, and 150 days, respectively, blood samples were taken 2ml. 样品储存于大约-70℃,直到分析。 Samples were stored at approximately -70 ℃, until analysis.

结果显示,所有治疗组均有血小板计数的升高,最大增值见于PEG-rHuMGDF和高剂量TMP组。 The results showed that all treatment groups had elevated platelet count, the maximum value found in the PEG-rHuMGDF and high dose TMP groups. 在第1周期,与对照组平均血小板计数相比,血小板计数峰值在PEG-rHuMGDF组大约增加3.3倍(第9天),在5000μg/kg TMP组大约增加3.1倍(第9天)。 In the first period, and compared to the control group mean platelet count, platelet count increase in peak of about 3.3 times the PEG-rHuMGDF group (Day 9), an increase of about 3.1 times in 5000μg / kg TMP group (Day 9). 在低剂量TMP组,在相同的特定研究时间,血小板计数较对照组增加约1.5倍。 In the low dose TMP group in the same study a particular time, platelet count significantly increased by about 1.5 times. 在所有其他周期中,可观察到相似反应。 In all other cycles, the similar reaction can be observed.

但是在第4周期,PEG-rHuMGDF组没有显示与前几个周期一样的血小板增量。 However, in the fourth cycle, PEG-rHuMGDF group did not show the same period the previous incremental platelet. 在这一周期给药后9天,PEG-rHuMGDF组血小板计数较对照组增加约2倍。 9 days after administration of the cycle, PEG-rHuMGDF group than the control group increased platelet counts of approximately 2-fold. 相对的,最高剂量TMP组在第4周期平均血小板计数比对照组增加约3.3倍。 In contrast, the highest dose TMP group in Cycle 4, mean platelet count increased by about 3.3 times than the control group. 另外,PEG-rHuMGDF组试验动物在第4周期开始时(每剂量),平均血小板计数较对照组平均血小板计数低53%,在第4周期结束时*(给药后27天),该组平均血小板计数较对照组低79%。 Additionally, PEG-rHuMGDF group of test animals at the beginning of the fourth period (per dose), mean platelet counts lower than the control group mean platelet count 53% * (27 days after administration), the average of the group at the end of cycle 4 platelet count 79% lower than the control group. 而对所有TMP试验动物而言,第4周期开始和结束时,其平均血小板计数是对照组的±15%。 For all TMP and experimental animals, the beginning and end of the fourth period, which is the mean platelet count ± 15% of the control group.

在第1和第2周期,所有治疗组对比对照组显示了红细胞(RBC)计数的下降趋势。 In the first and second period, all treatment groups compared to control group showed decreased red blood cell (RBC) counts. 下降在第41-43天最为显著,RBC最大的下降出现在PEG-rHuMGDF组。 At the most significant decline in 41-43 day, RBC largest decline in the PEG-rHuMGDF group. 早在第47天,计数开始回复到正常水平(与对照组相比)。 As early as day 47, the count began to return to normal levels (as compared to the control group). 白细胞(WBC)水平在第1和第2周期与对照组相比在第35天显著升高(2.6倍)。 White blood cell (WBC) levels in the first and second period compared with the control group increased significantly (2.6-fold) at 35 days. 在第33天,5000μg/kg TMP组显示了轻度升高。 On day 33, 5000μg / kg TMP group showed slightly increased. 从第37天开始,白细胞计数趋于正常(对照组)水平。 Starting on day 37, WBC count became normal (control) levels. 在第3周期观察到了相似的反应,而在第4周期,所有治疗组都没有明显变化。 In the third cycle similar response was observed, while in the fourth period, all treatment groups did not change significantly.

在第3周期,除了500μg/kgTMP组,所有治疗组在第13天(在第3周期单一剂量给药后)都显示了RBC计数的轻度下降。 In the third cycle, except that 500μg / kgTMP group, all treatment groups on day 13 (after the third cycle single dose) showed a slight drop in RBC counts. RBC计数在第17天开始回复正常水平(与对照组相比)。 RBC counts on day 17 to begin the return to normal levels (as compared to the control group).

在第4周期,除了500μg/kg TMP组,所有治疗组较对照组都显示了RBC下降。 In the fourth cycle, except that 500μg / kg TMP group, all treatment groups than in the control groups showed a decline RBC. 但与其他周期不同,在本周期中存在多个最低点。 But different from other cycles, there are multiple lowest point in this cycle. 这些下降从给药后1-9天开始显现,并在11天开始恢复。 The decline began to appear 1-9 days after the administration, 11 days and began to recover.

结果显示,在所有试验周期和所有试验动物中,给药7-9天后,可以检测到血小板计数较对照动物的升高。 The results show that in all the test period and all the test animals, 7-9 days after administration, the platelet count may be detected higher than that of control animals. 重复给药较之单剂给药可以显示更高的血小板产生反应。 Administration of a single dose repeated administration may be displayed higher than the platelets react. 在第4周期,PEG-rHuMGDF组诱导的血小板反应较之前几个周期要低,而且较高剂量TMP的反应也低。 In the fourth cycle, PEG-rHuMGDF group induced platelet effects than a few cycles prior to low and high dose TMP response is low. 在绝大多数治疗组和第1,2,3和4周期,在研究的每个周期的某些时间点,都观察到了RBC计数的下降,但是,在停药后,所有血液学参数都回复到正常水平(与对照组比较)。 In the vast majority of the treatment group and 2, 3 and 4 cycles, each cycle at certain time point of the study, the observed decrease in RBC count, however, after discontinuation, all replies are haematological parameters to normal levels (compared to control group).

总之,这些结果显示,恒河猴可以很好地耐受TMP治疗,而且TMP在多周期治疗后,都可增加血小板计数。 In summary, these results show that rhesus well tolerated treatment of TMP, and TMP after multiple cycles of treatment, may increase the platelet count. 基于血小板计数结果,没有显示针对TMP的显著生物免疫介导反应。 Based on the platelet count results, it did not show significant immune-mediated response against TMP. 相反,应用PEG-rHuMGDF治疗多个周期,在第4周期却显示了对血小板反应的抑制,提示机体产生了针对PEG-rHuMGDF的抗体,这些抗-MGDF抗体可能会与恒河猴内源性TPO发生交叉反应。 In contrast, application of PEG-rHuMGDF plurality of treatment cycles, the first cycle is 4 shows the inhibition of platelet response, suggesting that the body produces antibodies against PEG-rHuMGDF, which may be anti-antibodies within -MGDF endogenous rhesus TPO cross-react.

本发明现已充分描述,对于本领域的一般技术人员而言,在不背离这里公开的本发明的精神和范畴的情况下,可以做很多改变与修饰。 The present invention has been fully described, to those of ordinary skill in the art, the spirit and scope of the disclosure herein without departing from the present invention, many changes and modifications can be made.

序列表<110>Liu,Chuan-FaFeige,UlrichCheetham,Janet C. SEQUENCE LISTING & lt; 110 & gt; Liu, Chuan-FaFeige, UlrichCheetham, Janet C.

<120>血小板生成化合物<130>01017/36263<140> & Lt; 120 & gt; thrombopoietin compound & lt; 130 & gt; 01017/36263 & lt; 140 & gt;

<141> & Lt; 141 & gt;

<150>60/105,348<151>1998-10-23<160>46<170>PatentIn Ver.2.0<210>1<211>14<212>PRT<213>人工序列<220> & Lt; 150 & gt; 60 / 105,348 & lt; 151 & gt; 1998-10-23 & lt; 160 & gt; 46 & lt; 170 & gt; PatentIn Ver.2.0 & lt; 210 & gt; 1 & lt; 211 & gt; 14 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>1Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala1 5 10<210>2<211>14<212>PRT<213>人工序列 & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 1Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala1 5 10 & lt; 210 & gt; 2 & lt; 211 & gt; 14 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<220> & Lt; 223 & gt; Description of Artificial Sequence: peptide & lt; 220 & gt;

<223>肽是同型二聚体的亚单位:在每个二聚体亚单位的羧基末端都通过肽键与NH2-CH2-CH2CH2-CH2-CH(CONH2)-NH-CO-CH2-CH2-NH2共价连接。 & Lt; 223 & gt; peptide is a subunit of a homodimer: the carboxyl terminus of each subunit dimer through a peptide bond NH2-CH2-CH2CH2-CH2-CH (CONH2) -NH-CO-CH2CH2 -NH2 covalently linked.

<400>2Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala1 5 10<210>3<211>684<212>DNA<213>人工序列<220> & Lt; 400 & gt; 2Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala1 5 10 & lt; 210 & gt; 3 & lt; 211 & gt; 684 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>3atggacaaaa ctcacacatg tccaccttgt ccagctccgg aactcctggg gggaccgtca 60gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 240taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 300aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 360aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660agcctctccc tgtctccggg taaa 684<210>4<211>684<212>DNA<213>人工序列 agccctccca gcccccatcg 3atggacaaaa ctcacacatg tccaccttgt ccagctccgg aactcctggg gggaccgtca 60gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 180gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 240taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 300aagtgcaagg tctccaacaa; oligonucleotide & lt; 400 & gt: & lt;; 223 & gt artificial sequences described agaaaaccat ctccaaagcc 360aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 420aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660agcctctccc tgtctccggg taaa 684 & lt; 210 & gt; 4 & lt; 211 & gt; 684 & lt; 212 & gt; DNA & lt; 213 & gt ; artificial sequence

<220> & Lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>4tacctgtttt gagtgtgtac aggtggaaca ggtcgaggcc ttgaggaccc ccctggcagt 60cagaaggaga aggggggttt tgggttcctg tgggagtact agagggcctg gggactccag 120tgtacgcacc accacctgca ctcggtgctt ctgggactcc agttcaagtt gaccatgcac 180ctgccgcacc tccacgtatt acggttctgt ttcggcgccc tcctcgtcat gttgtcgtgc 240atggcacacc agtcgcagga gtggcaggac gtggtcctga ccgacttacc gttcctcatg 300ttcacgttcc agaggttgtt tcgggagggt cgggggtagc tcttttggta gaggtttcgg 360tttcccgtcg gggctcttgg tgtccacatg tgggacgggg gtagggccct actcgactgg 420ttcttggtcc agtcggactg gacggaccag tttccgaaga tagggtcgct gtagcggcac 480ctcaccctct cgttacccgt cggcctcttg ttgatgttct ggtgcggagg gcacgacctg 540aggctgccga ggaagaagga gatgtcgttc gagtggcacc tgttctcgtc caccgtcgtc 600cccttgcaga agagtacgag gcactacgta ctccgagacg tgttggtgat gtgcgtcttc 660tcggagaggg acagaggccc attt 684<210>5<211>228<212>PRT<213>人工序列<22 & Lt; 223 & gt; Artificial Sequence Description: Oligonucleotide & lt; 400 & gt; 4tacctgtttt gagtgtgtac aggtggaaca ggtcgaggcc ttgaggaccc ccctggcagt 60cagaaggaga aggggggttt tgggttcctg tgggagtact agagggcctg gggactccag 120tgtacgcacc accacctgca ctcggtgctt ctgggactcc agttcaagtt gaccatgcac 180ctgccgcacc tccacgtatt acggttctgt ttcggcgccc tcctcgtcat gttgtcgtgc 240atggcacacc agtcgcagga gtggcaggac gtggtcctga ccgacttacc gttcctcatg 300ttcacgttcc agaggttgtt tcgggagggt cgggggtagc tcttttggta gaggtttcgg 360tttcccgtcg gggctcttgg tgtccacatg tgggacgggg gtagggccct actcgactgg 420ttcttggtcc agtcggactg gacggaccag tttccgaaga tagggtcgct gtagcggcac 480ctcaccctct cgttacccgt cggcctcttg ttgatgttct ggtgcggagg gcacgacctg 540aggctgccga ggaagaagga gatgtcgttc gagtggcacc tgttctcgtc caccgtcgtc 600cccttgcaga agagtacgag gcactacgta ctccgagacg tgttggtgat gtgcgtcttc 660tcggagaggg acagaggccc attt 684 & lt; 210 & gt; 5 & lt; 211 & gt; 228 & lt; 212 & gt; PRT & lt; 213 & gt ; artificial sequence & lt; 22 0> 0 & gt;

<223>人工序列描述:肽<400>5Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60 & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 5Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gl n115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His G Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gl n115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His G lu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Gly Lys225<210>6<211>8<212>PRT<213>人工序列 lu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Gly Lys225 & lt; 210 & gt; 6 & lt; 211 & gt; 8 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>6Gly Gly Gly Lys Gly Gly Gly Gly1 5<210>7<211>8<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 6Gly Gly Gly Lys Gly Gly Gly Gly1 5 & lt; 210 & gt; 7 & lt; 211 & gt; 8 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>7Gly Gly Gly Asn Gly Ser Gly Gly1 5<210>8<211>8<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 7Gly Gly Gly Asn Gly Ser Gly Gly1 5 & lt; 210 & gt; 8 & lt; 211 & gt; 8 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>8Gly Gly Gly Cys Gly Gly Gly Gly1 5<210>9<211>4<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 8Gly Gly Gly Cys Gly Gly Gly Gly1 5 & lt; 210 & gt; 9 & lt; 211 & gt; 4 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽 & Lt; 223 & gt; Description of Artificial Sequence: Peptide

<400>9Gly Pro Asn Gly1<210>10<211>32<212>PRT<213>人工序列<220> & Lt; 400 & gt; 9Gly Pro Asn Gly1 & lt; 210 & gt; 10 & lt; 211 & gt; 32 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>10Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Pro1 5 10 15Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala20 25 30<210>11<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 10Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Pro1 5 10 15Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala20 25 30 & lt; 210 & gt; 11 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<220> & Lt; 223 & gt; Description of Artificial Sequence: peptide & lt; 220 & gt;

<223>环状肽;通过位于9和31位点的半胱氨酸残基间的分子内二硫键来维持二级结构。 & Lt; 223 & gt; cyclic peptide; secondary structure is maintained through 9 and 31 located at the site of an intramolecular disulfide bond between the cysteine ​​residues.

<400>11Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu20 25 30 & Lt; 400 & gt; 11Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu20 25 30

Ala Ala Arg Ala35<210>12<211>36<212>PRT<213>人工序列<220> Ala Ala Arg Ala35 & lt; 210 & gt; 12 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>12Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Tle Glu Gly Pro Thr Arg Leu Gln Cys Leu20 25 30Ala Ala Arg Ala35<210>13<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 12Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Tle Glu Gly Pro Thr Arg Leu Gln Cys Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 13 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>13Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu20 25 30Ala Ala Arg Ala35 & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 13Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu20 25 30Ala Ala Arg Ala35

<210>14<211>36<212>PRT<213>人工序列<220> & Lt; 210 & gt; 14 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>14Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>15<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 14Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 15 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>位于18位点的赖氨酸残基溴乙酰化。 & Lt; 223 & gt; located at 18 points bromoacetylated lysine residue.

<220> & Lt; 220 & gt;

<223>人工序列描述:衍生肽<400>15Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35 & Lt; 223 & gt; Artificial Sequence Description: derived peptide & lt; 400 & gt; 15Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35

<210>16<211>36<212>PRT<213>人工序列<220> & Lt; 210 & gt; 16 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>16Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>17<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 16Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 17 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>位于18位点的赖氨酸聚乙烯二醇化。 & Lt; 223 & gt; located at 18 points pegylated lysine.

<220> & Lt; 220 & gt;

<223>人工序列描述:衍生肽<400>17Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35 & Lt; 223 & gt; Artificial Sequence Description: derived peptide & lt; 400 & gt; 17Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35

<210>18<211>36<212>PRT<213>人工序列<220> & Lt; 210 & gt; 18 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>位于18位点的半胱氨酸聚乙烯二醇化。 & Lt; 223 & gt; site is located 18 pegylated cysteine.

<220> & Lt; 220 & gt;

<223>人工序列描述:衍生肽<400>18Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>19<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Sequence Description: derived peptide & lt; 400 & gt; 18Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala ala Arg Ala35 & lt; 210 & gt; 19 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>19Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Asn Gly Ser Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35 & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 19Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Asn Gly Ser Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35

<210>20<211>36<212>PRT<213>人工序列<220> & Lt; 210 & gt; 20 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>同型二聚体的单体亚单位;同型二聚体中的亚单位通过位于每个亚单位18位点的半胱氨酸残基间二硫键连接。 & Lt; 223 & gt; homodimer monomeric subunits; homodimers of subunits disulfide bonded between cysteine ​​residues located on each subunit by 18 points.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>20Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>21<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 20Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 21 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>21Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 21Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala

35<210>22<211>32<212>PRT<213>人工序列<220> 35 & lt; 210 & gt; 22 & lt; 211 & gt; 32 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>通过在肽氨基末端共价连接免疫球蛋白Fc段来衍生肽。 & Lt; 223 & gt; connected by a peptide derivatized immunoglobulin Fc amino terminus of the peptide segment is covalently.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>22Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Pro1 5 10 15Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala20 25 30<210>23<211>32<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 22Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Pro1 5 10 15Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala20 25 30 & lt; 210 & gt; 23 & lt; 211 & gt; 32 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>肽的氨基和羧基末端均与一个免疫球蛋白Fc段共价连接。 & Lt; 223 & gt; peptide amino and carboxy termini are connected covalently to a Fc fragment of an immunoglobulin.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>23Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Pro1 5 10 15Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala20 25 30 & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 23Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Pro1 5 10 15Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala20 25 30

<210>24<211>36<212>PRT<213>人下序列<220> & Lt; 210 & gt; 24 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; the human sequence & lt; 220 & gt;

<223>肽的羧基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently carboxy terminus of the immunoglobulin peptide connection.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>24Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>25<211>34<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 24Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 25 & lt; 211 & gt; 34 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>25Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala1 5 10 15Gly Pro Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala20 25 30 & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 25Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala1 5 10 15Gly Pro Asn Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala20 25 30

Arg Ala<210>26<211>36<212>PRT<213>人工序列<220> Arg Ala & lt; 210 & gt; 26 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>26Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>27<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 26Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 27 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<220> & Lt; 220 & gt;

<223>环状肽;通过位于9和31位点的半胱氨酸残基间的分子内二硫键来维持二级结构。 & Lt; 223 & gt; cyclic peptide; secondary structure is maintained through 9 and 31 located at the site of an intramolecular disulfide bond between the cysteine ​​residues.

<220> & Lt; 220 & gt;

<223>人工序列描述;肽<400>27Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly & Lt; 223 & gt; Description of artificial sequence; peptide & lt; 400 & gt; 27Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly

1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu20 25 30Ala Ala Arg Ala35<210>28<211>36<212>PRT<213>人工序列<220> 1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu20 25 30Ala Ala Arg Ala35 & lt; 210 & gt; 28 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>28Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu20 25 30Ala Ala Arg Ala35<210>29<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 28Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 29 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<220> & Lt; 223 & gt; Description of Artificial Sequence: peptide & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<400>29Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu20 25 30Ala Ala Arg Ala35<210>30<211>36<212>PRT<213>人工序列<220> & Lt; 400 & gt; 29Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Ala Leu20 25 30Ala Ala Arg Ala35 & lt; 210 & gt; 30 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>30Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>31<211>36<212>PRT<213>人工序列 & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 30Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 31 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence

<220> & Lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<220> & Lt; 220 & gt;

<223>人工序列描述:肽<400>31Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>32<211>36<212>PRT<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 31Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala arg Ala35 & lt; 210 & gt; 32 & lt; 211 & gt; 36 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<220> & Lt; 223 & gt; Description of Artificial Sequence: peptide & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<400>32Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Asn Gly Ser Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>33<211>36 & Lt; 400 & gt; 32Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Asn Gly Ser Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35 & lt; 210 & gt; 33 & lt; 211 & gt; 36

<212>PRT<213>人工序列<220> & Lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<220> & Lt; 223 & gt; Description of Artificial Sequence: peptide & lt; 220 & gt;

<223>肽是同型二聚体的亚单位;同型二聚体中的亚单位通过位于每个亚单位18位点的半胱氨酸残基间二硫键连接。 & Lt; 223 & gt; peptide is a subunit of a homodimer; homodimers of subunits disulfide bonded between cysteine ​​residues located on each subunit by 18 points.

<220> & Lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<400>33Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35<210>34<211>41<212>PRT<213>人工序列<220> & Lt; 400 & gt; 33Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly1 5 10 15Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu20 25 30Ala Ala Arg Ala35 & lt; 210 & gt; 34 & lt; 211 & gt; 41 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<220> & Lt; 223 & gt; Description of Artificial Sequence: peptide & lt; 220 & gt;

<223>肽的氨基末端与免疫球蛋白的Fc段共价连接。 & Lt; 223 & gt; Fc segment covalently amino terminus of an immunoglobulin peptide is attached.

<400>34Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala1 5 10 15 & Lt; 400 & gt; 34Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala1 5 10 15

Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr20 25 30Leu Arg Gln Trp Leu Ala Ala Arg Ala35 40<210>35<211>60<212>DNA<213>人工序列<220> Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr20 25 30Leu Arg Gln Trp Leu Ala Ala Arg Ala35 40 & lt; 210 & gt; 35 & lt; 211 & gt; 60 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>35aaaggtggag gtggtggtat cgaaggtccg actctgcgtc agtggctggc tgctcgtgct 60<210>36<211>48<212>DNA<213>人工序列<220> & Lt; 223 & gt; Artificial Sequence Description: Oligonucleotide & lt; 400 & gt; 35aaaggtggag gtggtggtat cgaaggtccg actctgcgtc agtggctggc tgctcgtgct 60 & lt; 210 & gt; 36 & lt; 211 & gt; 48 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>36acctccacca ccagcacgag cagccagcca ctgacgcaga gtcggacc 48<210>37<211>66<212>DNA<213>人工序列<220> & Lt; 223 & gt; Artificial Sequence Description: Oligonucleotide & lt; 400 & gt; 36acctccacca ccagcacgag cagccagcca ctgacgcaga gtcggacc 48 & lt; 210 & gt; 37 & lt; 211 & gt; 66 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸 & Lt; 223 & gt; Description of Artificial Sequence: Oligonucleotide

<400>37ggtggtggag gtggcggcgg aggtattgag ggcccaaccc ttcgccaatg gcttgcagca 60cgcgca 66<210>38<211>76<212>DNA<213>人工序列<220> & Lt; 400 & gt; 37ggtggtggag gtggcggcgg aggtattgag ggcccaaccc ttcgccaatg gcttgcagca 60cgcgca 66 & lt; 210 & gt; 38 & lt; 211 & gt; 76 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>38aaaaaaagga tcctcgagat tatgcgcgtg ctgcaagcca ttggcgaagg gttgggccct 60caatacctcc gccgcc 76<210>39<211>126<212>DNA<213>人工序列<220> & Lt; 223 & gt; Artificial Sequence Description: Oligonucleotide & lt; 400 & gt; 38aaaaaaagga tcctcgagat tatgcgcgtg ctgcaagcca ttggcgaagg gttgggccct 60caatacctcc gccgcc 76 & lt; 210 & gt; 39 & lt; 211 & gt; 126 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>39aaaggtggag gtggtggtat cgaaggtccg actctgcgtc agtggctggc tgctcgtgct 60ggtggtggag gtggcggcgg aggtattgag ggcccaaccc ttcgccaatg gcttgcagca 120cgcgca 126<210>40<211>124<212>DNA<213>人工序列<220> & Lt; 223 & gt; Artificial Sequence Description: Oligonucleotide & lt; 400 & gt; 39aaaggtggag gtggtggtat cgaaggtccg actctgcgtc agtggctggc tgctcgtgct 60ggtggtggag gtggcggcgg aggtattgag ggcccaaccc ttcgccaatg gcttgcagca 120cgcgca 126 & lt; 210 & gt; 40 & lt; 211 & gt; 124 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt ;

<223>人工序列描述:寡核苷酸 & Lt; 223 & gt; Description of Artificial Sequence: Oligonucleotide

<400>40ccaggctgag acgcagtcac cgaccgacga gcacgaccac cacctccacc gccgcctcca 60taactcccgg gttgggaagc ggttaccgaa cgtcgtgcgc gtattagagc tcctaggaaa 120aaaa 124<210>41<211>42<212>PRT<213>人工序列<220> & Lt; 400 & gt; 40ccaggctgag acgcagtcac cgaccgacga gcacgaccac cacctccacc gccgcctcca 60taactcccgg gttgggaagc ggttaccgaa cgtcgtgcgc gtattagagc tcctaggaaa 120aaaa 124 & lt; 210 & gt; 41 & lt; 211 & gt; 42 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>41Lys Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15Ala Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro20 25 30Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala35 40<210>42<211>22<212>DNA<213>人工序列<220> & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 41Lys Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15Ala Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro20 25 30Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala35 40 & lt; 210 & gt; 42 & lt; 211 & gt; 22 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>42aacataagta cctgtaggat cg 22<210>43<211>52 & Lt; 223 & gt; Description of Artificial Sequence: Oligonucleotide & lt; 400 & gt; 42aacataagta cctgtaggat cg 22 & lt; 210 & gt; 43 & lt; 211 & gt; 52

<212>DNA<213>人工序列<220> & Lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>43ttcgatacca ccacctccac ctttacccgg agacagggag aggctcttct gc 52<210>44<211>861<212>DNA<213>人工序列<220> & Lt; 223 & gt; Artificial Sequence Description: Oligonucleotide & lt; 400 & gt; 43ttcgatacca ccacctccac ctttacccgg agacagggag aggctcttct gc 52 & lt; 210 & gt; 44 & lt; 211 & gt; 861 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>44tctagatttg ttttaactaa ttaaaggagg aataacatat ggacaaaact cacacatgtc 60caccttgtcc agctccggaa ctcctggggg gaccgtcagt cttcctcttc cccccaaaac 120ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga 180gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag gtgcataatg 240ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc agcgtcctca 300ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag 360ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac 420aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc agcctgacct 480gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc 540cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc ttcttcctct 600acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg 660tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta 720aaggtggagg tggtggtatc gaaggtccga ctctgcgtca gtggctggct gctcgtgctg ctgaatggca aggagtacaa 44tctagatttg ttttaactaa ttaaaggagg aataacatat ggacaaaact cacacatgtc 60caccttgtcc agctccggaa ctcctggggg gaccgtcagt cttcctcttc cccccaaaac 120ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga 180gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag gtgcataatg 240ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc agcgtcctca 300ccgtcctgca ccaggactgg; oligonucleotide & lt; 400 & gt: & lt;; 223 & gt artificial sequences described gtgcaaggtc tccaacaaag 360ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac 420aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc agcctgacct 480gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc 540cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc ttcttcctct 600acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg 660tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta 720aaggtggagg tggtggtatc gaaggtccga ctctgcgtca gtggctggct gctcgtgctg 780gtggtggagg tggcggcgga ggtattgagg gcccaaccct tcgccaatgg cttgcagcac 840gcgcataatc tcgaggatcc g 861<210>45<211>861<212>DNA 780gtggtggagg tggcggcgga ggtattgagg gcccaaccct tcgccaatgg cttgcagcac 840gcgcataatc tcgaggatcc g 861 & lt; 210 & gt; 45 & lt; 211 & gt; 861 & lt; 212 & gt; DNA

<213>人工序列<220> & Lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:寡核苷酸<400>45agatctaaac aaaattgatt aatttcctcc ttattgtata cctgttttga gtgtgtacag 60gtggaacagg tcgaggcctt gaggaccccc ctggcagtca gaaggagaag gggggttttg 120ggttcctgtg ggagtactag agggcctggg gactccagtg tacgcaccac cacctgcact 180cggtgcttct gggactccag ttcaagttga ccatgcacct gccgcacctc cacgtattac 240ggttctgttt cggcgccctc ctcgtcatgt tgtcgtgcat ggcacaccag tcgcaggagt 300ggcaggacgt ggtcctgacc gacttaccgt tcctcatgtt cacgttccag aggttgtttc 360gggagggtcg ggggtagctc ttttggtaga ggtttcggtt tcccgtcggg gctcttggtg 420tccacatgtg ggacgggggt agggccctac tcgactggtt cttggtccag tcggactgga 480cggaccagtt tccgaagata gggtcgctgt agcggcacct caccctctcg ttacccgtcg 540gcctcttgtt gatgttctgg tgcggagggc acgacctgag gctgccgagg aagaaggaga 600tgtcgttcga gtggcacctg ttctcgtcca ccgtcgtccc cttgcagaag agtacgaggc 660actacgtact ccgagacgtg ttggtgatgt gcgtcttctc ggagagggac agaggcccat 720ttccacctcc accaccatag cttccaggct gagacgcagt caccgaccga cgagcacgac gacttaccgt tcctcatgtt 45agatctaaac aaaattgatt aatttcctcc ttattgtata cctgttttga gtgtgtacag 60gtggaacagg tcgaggcctt gaggaccccc ctggcagtca gaaggagaag gggggttttg 120ggttcctgtg ggagtactag agggcctggg gactccagtg tacgcaccac cacctgcact 180cggtgcttct gggactccag ttcaagttga ccatgcacct gccgcacctc cacgtattac 240ggttctgttt cggcgccctc ctcgtcatgt tgtcgtgcat ggcacaccag tcgcaggagt 300ggcaggacgt ggtcctgacc; oligonucleotide & lt; 400 & gt: & lt;; 223 & gt artificial sequences described cacgttccag aggttgtttc 360gggagggtcg ggggtagctc ttttggtaga ggtttcggtt tcccgtcggg gctcttggtg 420tccacatgtg ggacgggggt agggccctac tcgactggtt cttggtccag tcggactgga 480cggaccagtt tccgaagata gggtcgctgt agcggcacct caccctctcg ttacccgtcg 540gcctcttgtt gatgttctgg tgcggagggc acgacctgag gctgccgagg aagaaggaga 600tgtcgttcga gtggcacctg ttctcgtcca ccgtcgtccc cttgcagaag agtacgaggc 660actacgtact ccgagacgtg ttggtgatgt gcgtcttctc ggagagggac agaggcccat 720ttccacctcc accaccatag cttccaggct gagacgcagt caccgaccga cgagcacgac 780caccacctcc accgccgcct ccataactcc cgggttggga agcggttacc gaacgtcgtg 840cgcgtattag agctcctagg c 861<210>46<211>269<212>PRT<213>人工序列<220> 780caccacctcc accgccgcct ccataactcc cgggttggga agcggttacc gaacgtcgtg 840cgcgtattag agctcctagg c 861 & lt; 210 & gt; 46 & lt; 211 & gt; 269 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>人工序列描述:肽<400>46Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser & Lt; 223 & gt; Artificial Description: Peptide & lt; 400 & gt; 46Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp trp Leu Asn85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Gly Lys Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg225 230 235 240Gln Trp Leu Ala Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Gly Lys Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg225 230 235 240Gln Trp Leu Ala Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile

245 250 255Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala260 265 245 250 255Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala260 265

Claims (43)

1.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4是脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 1. The compounds of the following structure comprising mpl receptor binding TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group comprising a compound of the following core structure: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, lysine and valine; X3 is selected from glycine and alanine; X4 is proline; X5 is selected from threonine and serine; X6 is selected from light leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine, asparagine and glutamic acid; X9 is selected from tryptophan, tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine and lysine acid; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
2.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3是丙氨酸;X4是脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 2. A compound comprising the following structure mpl receptor binding TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group of core compounds comprising the structure: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is alanine; X4 is proline; X5 is selected from threonine and serine; X6 is selected from from leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine, asparagine and glutamic acid; X9 It is selected from tryptophan, tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine and lysine; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
3.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4是脯氨酸;X5是丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 3. A compound comprising the following structure mpl receptor binding TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group comprising a compound of the following core structure: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is proline; X5 is a serine; X6 is selected from light leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine, asparagine and glutamic acid; X9 is selected from tryptophan, tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine and lysine acid; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
4.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4是脯氨酸;X5选自苏氨酸和丝氨酸;X6选自异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 4. A compound comprising the following structure mpl receptor binding TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group of core compounds comprising the structure: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is proline; X5 is selected from threonine and serine ; X6 is selected from isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine, asparagine and glutamic acid; X9 is selected from since tryptophan, tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine, and lysine; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
5.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4是脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7是赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 5. A compound comprising the following structure mpl receptor binding TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group comprising a compound of the following core structure: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is proline; X5 is selected from threonine and serine ; X6 is selected from leucine, isoleucine, valine, alanine and phenylalanine; X7 is lysine; X8 is selected from glutamine, asparagine and glutamic acid; X9 is selected from tryptophan, tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine and lysine acid; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
6.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4是脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺和天门冬酰胺;X9选自色氨酸、酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 6. A compound comprising the following structure mpl receptor binding TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group comprising a compound of the following core structure: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is proline; X5 is selected from threonine and serine ; X6 is selected from leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine and asparagine; X9 is selected from since tryptophan, tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine, and lysine; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
7.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4是脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 7. A compound comprising TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group comprising a compound with the following core structure of mpl receptor binding: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is proline; X5 is selected from threonine and serine ; X6 is selected from leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine, asparagine and glutamine acid; X9 is selected from tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, isoleucine, valine, alanine, phenylalanine, methionine, and lysine; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
8.包括下述结构与mpl受体结合的化合物TMP1-(L1)n-TMP2其中TMP1和TMP2分别独立选自包括以下结构的一组核心化合物:X2-X3-X4-X5-X6-X7-X8-X9-X10其中,X2选自谷氨酸、天门冬氨酸、赖氨酸和缬氨酸;X3选自甘氨酸和丙氨酸;X4选自脯氨酸;X5选自苏氨酸和丝氨酸;X6选自亮氨酸、异亮氨酸、缬氨酸、丙氨酸和苯丙氨酸;X7选自精氨酸和赖氨酸;X8选自谷氨酰胺、天门冬酰胺和谷氨酸;X9选自色氨酸、酪氨酸、半胱氨酸、丙氨酸和苯丙氨酸;X10选自亮氨酸、缬氨酸、丙氨酸、苯丙氨酸、蛋氨酸和赖氨酸;L1为接头;并且n是0或1;及其生理学可以接受的盐类。 8. A compound comprising TMP1- (L1) n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group comprising a compound with the following core structure of mpl receptor binding: X2-X3-X4-X5-X6-X7- wherein X8-X9-X10, an X2 is selected from glutamic acid, aspartic acid, lysine, and valine; X3 is selected from glycine and alanine; X4 is selected from proline; and X5 is selected from threonine serine; X6 is selected from leucine, isoleucine, valine, alanine and phenylalanine; X7 is selected from arginine and lysine; X8 is selected from glutamine, asparagine and valleys acid; X9 is selected from tryptophan, tyrosine, cysteine, alanine and phenylalanine; X10 is selected from leucine, valine, alanine, phenylalanine, methionine, and lysine; Ll is a linker; and n is 0 or 1; and their physiologically acceptable salts thereof.
9.根据权利要求1-8中任一项的化合物,其中TMP1和TMP2独立选自:X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X2-X3-X4-×5-X6-X7-X8-X9-X10-X11-X12-X13;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13;和X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14,其中X2-X10如定义;X1选自异亮氨酸、缬氨酸、亮氨酸、丝氨酸和精氨酸;X11选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、赖氨酸、组氨酸和谷氨酸;X12选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、甘氨酸、丝氨酸和谷氨酰胺;X13选自精氨酸、赖氨酸、苏氨酸、缬氨酸、天门冬酰胺、谷氨酰胺和甘氨酸;并且X14选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、苏氨酸、精氨酸 9. A compound according to any one of claims 1-8, wherein TMP1 and TMP2 are independently selected from: X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; X2-X3-X4-X5 -X6-X7-X8-X9-X10-X11-X12; X2-X3-X4- × 5-X6-X7-X8-X9-X10-X11-X12-X13; X2-X3-X4-X5-X6- X7-X8-X9-X10-X11-X12-X13-X14; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10; X1-X2-X3-X4-X5-X6-X7- X8-X9-X10-X11; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12; X1-X2-X3-X4-X5-X6-X7-X8-X9- X10-X11-X12-X13; and X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein X2-X10 are as defined; the X1 is selected from isoleucine acid, valine, leucine, serine, and arginine; X11 is selected from alanine, isoleucine, valine, leucine, phenylalanine, serine, threonine, lysine , histidine and glutamic acid; X12 is selected from alanine, isoleucine, valine, leucine, phenylalanine, glycine, serine and glutamine; X13 is selected from arginine, lysine , threonine, valine, asparagine, glutamine, and glycine; and X14 is selected from alanine, isoleucine, valine, leucine, phenylalanine, threonine arginine 谷氨酸和甘氨酸。 Glutamate and glycine.
10.根据权利要求1-8中任一项的化合物,其中TMP1和TMP2独立选自:X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13;和X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14,其中X2-X10如定义;X1选自异亮氨酸、丙氨酸、缬氨酸、亮氨酸、丝氨酸和精氨酸;X11选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、苏氨酸、赖氨酸、组氨酸和谷氨酸;X12选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、甘氨酸、丝氨酸和谷氨酰胺;X13选自精氨酸、赖氨酸、苏氨酸、缬氨酸、天门冬酰胺、谷氨酰胺和甘氨酸;并且X14选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、苏氨酸、精氨酸 10. A compound as claimed in any one of the claims, wherein TMP1 and TMP2 are independently selected from: X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; X2-X3-X4-X5 -X6-X7-X8-X9-X10-X11-X12; X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13; X2-X3-X4-X5-X6-X7 -X8-X9-X10-X11-X12-X13-X14; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10; X1-X2-X3-X4-X5-X6-X7-X8 -X9-X10-X11; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 -X11-X12-X13; and X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein X2-X10 are as defined; the X1 is selected from isoleucine , alanine, valine, leucine, serine, and arginine; X11 is selected from alanine, isoleucine, valine, leucine, phenylalanine, threonine, lysine acid, histidine, and glutamic acid; X12 is selected from alanine, isoleucine, valine, leucine, phenylalanine, glycine, serine and glutamine; X13 is selected from arginine, lysine, threonine, valine, asparagine, glutamine, and glycine; and X14 is selected from alanine, isoleucine, valine, leucine, phenylalanine, threonine methyl acid, arginine 谷氨酸和甘氨酸。 Glutamate and glycine.
11.根据权利要求1-8中任一项的化合物,其中TMP1和TMP2独立选自:X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13;X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12;X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13;和X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14,其中X2-X10如定义;X1选自异亮氨酸、丙氨酸、缬氨酸、亮氨酸、丝氨酸和精氨酸;X11选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、赖氨酸、组氨酸和谷氨酸;X12选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、甘氨酸、丝氨酸和谷氨酰胺;X13选自精氨酸、赖氨酸、苏氨酸、缬氨酸、天门冬酰胺、谷氨酰胺和甘氨酸;并且X14选自丙氨酸、异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、苏氨酸、精氨酸、 11. A compound as claimed in any one of the claims, wherein TMP1 and TMP2 are independently selected from: X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; X2-X3-X4-X5 -X6-X7-X8-X9-X10-X11-X12; X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13; X2-X3-X4-X5-X6-X7 -X8-X9-X10-X11-X12-X13-X14; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10; X1-X2-X3-X4-X5-X6-X7-X8 -X9-X10-X11; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 -X11-X12-X13; and X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein X2-X10 are as defined; the X1 is selected from isoleucine , alanine, valine, leucine, serine, and arginine; X11 is selected from alanine, isoleucine, valine, leucine, phenylalanine, serine, threonine, lysine, histidine and glutamic acid; X12 is selected from alanine, isoleucine, valine, leucine, glycine, serine and glutamine; X13 is selected from arginine, lysine , threonine, valine, asparagine, glutamine, and glycine; and X14 is selected from alanine, isoleucine, valine, leucine, phenylalanine, threonine, fine acid, 氨酸和甘氨酸。 Histidine and glycine.
12.根据权利要求1-8中任一项的化合物,其中TMP1和/或TMP2是包括如下所示的一种或多种修饰的衍生物:一个或多个肽[-C(O)NR-]键被非肽键所取代,该非肽键选自-CH2-氨甲酸酯键[-CH2-OC(O)NR-];膦酸酯键;-CH2-磺胺[-CH2-S(O)2NR-]键;脲[-NHC(O)NH-]键;-CH2-仲胺键;和烷基化的肽键[-C(O)NR6-其中R6是低级烷基];N末端为一个-NRR1基团;为一个-NRC(O)R基团;为一个-NRC(O)OR基团;为一个-NRS(O)2R基团;为一个-NHC(O)NHR基团,其中R和R1是氢原子或低级烷基,但条件是R和R1不能同时为氢原子;为一个琥珀酰亚胺基团;为一个苄氧羰基-NH-(CBZ-NH-)基团;或一个苄氧羰基-NH-基团,其中苯环有1-3个取代物,分别选自低级烷基、低级烷氧基、氯和溴;C末端为-C(O)R2,其中R2选自低级烷氧基,以及-NR3R4,其中R3和R4独立选自氢原子和低级烷基。 12. A compound as claimed in any one of the claims, wherein TMP1 and / or TMP2 are shown below comprising one or more modified derivatives: one or more peptide [-C (O) NR- ] keys are replaced by non-peptide bonds, the non-peptide bond selected from -CH2- urethane bond [-CH2-OC (O) NR-]; phosphonate bond; -CH2- sulfonamide [-CH2-S ( O) 2NR-] linkage; urea [-NHC (O) NH-] linkage; -CH2- secondary amine linkage; and alkylated peptide bond [-C (O) NR6- wherein R6 is lower alkyl]; N end of a -NRR1 group; a -NRC (O) R group; to a -NRC (O) OR group; a -NRS (O) 2R group; a -NHC (O) NHR group group, wherein R and R1 is a hydrogen atom or a lower alkyl group, with the proviso that R and R1 are not simultaneously a hydrogen atom; a succinimide group; a benzyloxycarbonyl group -NH- (CBZ-NH-) group group; or a benzyloxycarbonyl group -NH- group, wherein the phenyl ring with 1-3 substituents, independently selected from lower alkyl, lower alkoxy, chlorine and bromine; C-terminus is -C (O) R2, wherein R2 is selected from lower alkoxy, and -NR3R4, wherein R3 and R4 are independently selected from hydrogen atoms and lower alkyl.
13.根据权利要求1-8中任一项的化合物,其中所有的氨基酸都是D构型。 13. A compound as claimed in any one of the claims, wherein all amino acids are in the D configuration.
14.根据权利要求1-8中任一项的化合物,其中至少一个氨基酸是D构型。 14. The compound as claimed in any one of the claims, wherein the at least one amino acid in D configuration.
15.根据权利要求1-8中任一项的化合物,为环状。 15. A compound as claimed in any one of the claims, a ring shape.
16.根据权利要求1-8中任一项的化合物,其中TMP1和TMP2都是序列1:Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala。 16. A compound as claimed in any one of the claims, wherein TMP1 and TMP2 are sequence 1: Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg- Ala.
17.根据权利要求1-8中任一项的化合物,其中L1包括一种肽。 17. A compound as claimed in any one of the claims, wherein L1 comprises a peptide.
18.根据权利要求17的化合物,其中L1包括Yn,其中Y是天然氨基酸或其立体异构体,n是1-20的数字。 18. A compound according to claim 17, wherein L1 comprises Yn of wherein Y is a natural amino acid or a stereoisomer thereof, n being numbers from 1 to 20.
19.根据权利要求17的化合物,其中L1包括(甘氨酸)n,其中n是1-20的数字,并且当n大于1时,高达半数的甘氨酸残基可以被选自剩余19种天然氨基酸或其立体异构体的氨基酸取代。 19. The compound according to claim 17, wherein L1 comprises (Gly) n, where n is a number from 1 to 20, and when n is greater than 1, up to half of the Gly residues may be selected from the remaining 19 kinds of natural amino acids, or substituted amino acid stereoisomers.
20.根据权利要求17的化合物,其中L1选自:序列6:(甘氨酸)3赖氨酸(甘氨酸)4;序列7:(甘氨酸)3天门冬酰胺甘氨酸丝氨酸(甘氨酸)2;序列8:(甘氨酸)3半胱氨酸(甘氨酸)4;以及序列9:甘氨酸脯氨酸天门冬酰胺甘氨酸。 20. The compound according to claim 17, wherein L1 is selected from: 6 :( sequence Gly) 3 Lys (Gly) 4; 7 :( sequence Gly) 3 Ser asparagine glycine (Gly) 2; 8 sequences :( Gly) 3 Cys (Gly) 4; and sequence 9: glycine, proline, asparagine glycine.
21.根据权利要求17的化合物,其中L1包括一个半胱氨酸残基。 21. The compound according to claim 17, wherein L1 comprises a cysteine ​​residue.
22.根据权利要求21的化合物的二聚体。 22. A dimer of the compound according to claim 21.
23.根据权利要求22的二聚体,其为TMP1-Gly3-Cys-Gly4-TMP2|TMP1-Gly3-Cys-Gly4-TMP2. 23. Dimer according to claim 22, which is TMP1-Gly3-Cys-Gly4-TMP2 | TMP1-Gly3-Cys-Gly4-TMP2.
24.根据权利要求1-8中任一项的化合物,其中L1包括(CH2)n,其中n是1-20的数字。 24. A compound as claimed in any one of the claims, wherein L1 comprises (the CH2) n, where n is a number from 1 to 20.
25.根据权利要求1-8中任一项的化合物,选自:序列10:IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA序列11:IEGPTLRQCLAARA-GGGGGGGG-IEPTLRQCLAARA,其为环状 25. A compound as claimed in any one of the claim 1 selected from: SEQ ID 10: IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA sequence 11: IEGPTLRQCLAARA-GGGGGGGG-IEPTLRQCLAARA, which is a cyclic 序列12:IEGPTLRQCLAARA-GGGGGGG-IEGPTLRQCLAARA,其为线性序列13:IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA序列14:IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA序列15:IEGPTLRQWLAARA-GGGK(BrAc)GGGG-IEGPTLRQWLAARA序列16:IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA序列17:IEGPTLRQWLAARA-GGGK(PEG)GGGG-IEGPTLRQWLAARA序列18:IEGPTLRQWLAARA-GGGC(PEG)GGGG-IEGPTLRQWLAARA序列19:IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA序列20:IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA|IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA序列21:IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA。 Sequence 12: IEGPTLRQCLAARA-GGGGGGG-IEGPTLRQCLAARA, which is a linear sequence of 13: IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA sequence 14: IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA sequence 15: IEGPTLRQWLAARA-GGGK (BrAc) GGGG-IEGPTLRQWLAARA sequence 16: IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA sequence 17 : IEGPTLRQWLAARA-GGGK (PEG) GGGG-IEGPTLRQWLAARA sequence 18: IEGPTLRQWLAARA-GGGC (PEG) GGGG-IEGPTLRQWLAARA sequence 19: IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA sequence 20: IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA | IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA sequence 21: IEGPTLRQWLAARA-GGGGGGGG -IEGPTLRQWLAARA.
26.根据权利要求1-11中任一项的化合物,具有以下结构式(Fc)m-(L2)q-TMP1-(L1)n-TMP2-(L3)r-(Fc)p其中,L1,L2和L3是分别独立选自以下接头基团的接头基团:Yn,其中Y是天然氨基酸或其立体异构体,n是1-20的数字;(甘氨酸)n,其中n是1-20的数字,并且当n大于1时,高达半数的甘氨酸残基可以被选自剩余19种天然氨基酸或其立体异构体的氨基酸取代;序列6:(甘氨酸)3赖氨酸(甘氨酸)4;序列7:(甘氨酸)3天门冬酰胺甘氨酸丝氨酸(甘氨酸)2;序列8:(甘氨酸)3半胱氨酸(甘氨酸)4;序列9:甘氨酸脯氨酸天门冬酰胺甘氨酸;半胱氨酸残基;以及(CH2)n,其中n是1-20的数字;Fc是免疫球蛋白的Fc段;m,p,q和r是分别独立选自0和1的数字,其中m或p至少有一个是1,而且,如果m为0则q为0,如果p为0则r为0;及其生理学可以接受的盐。 26. A compound according to any one of claims 1-11 claim, having the structure of formula (Fc) m- (L2) q-TMP1- (L1) n-TMP2- (L3) r- (Fc) p wherein, L1, L2 and L3 are each independently selected from the linker group of the following linker groups: Yn, wherein Y is a natural amino acid or a stereoisomer thereof, n being numbers from 1 to 20; (Gly) n, where n is 1-20 numbers, and when n is greater than 1, up to half of the Gly residues may be selected from the remaining 19 kinds of natural amino acids or a substitution of stereoisomers; 6 :( sequence Gly) 3 Lys (Gly) 4; 7 :( sequence Gly) 3 Ser asparagine glycine (Gly) 2; 8 :( sequence Gly) 3 Cys (Gly) 4; SEQ ID 9: proline, glycine asparagine glycine; cysteine ​​residues group; and (the CH2) n, where n is a number from 1 to 20; Fc is the Fc portion of an immunoglobulin; m, p, Q and r are each independently selected from numbers 0 and 1, wherein at least m or p a is 1, and, if m is 0 then q is 0, p is 0 if r is 0; and physiologically acceptable salts thereof.
27.根据权利要求26的化合物,其中L1,L2和L3分别独立选自Yn,其中Y是天然氨基酸或其立体异构体,n是1-20的数字。 27. The compound according to claim 26, wherein L1, L2 and L3 are each independently selected Yn of wherein Y is a natural amino acid or a stereoisomer thereof, n being numbers from 1 to 20.
28.根据权利要求27的化合物,其中L1含有(甘氨酸)n,其中n是1-20的数字,并且当n大于1时,高达半数的甘氨酸残基可以被选自剩余19种天然氨基酸或其立体异构体的氨基酸取代。 28. The compound according to claim 27, wherein L1 comprises (Gly) n, where n is a number from 1 to 20, and when n is greater than 1, up to half of the Gly residues may be selected from the remaining 19 kinds of natural amino acids, or substituted amino acid stereoisomers.
29.根据权利要求27的化合物,其中L1,L2和L3分别独立选自:序列6:(甘氨酸)3赖氨酸(甘氨酸)4;序列7:(甘氨酸)3天门冬酰胺甘氨酸丝氨酸(甘氨酸)2;序列8:(甘氨酸)3半胱氨酸(甘氨酸)4;以及序列9:甘氨酸脯氨酸天门冬酰胺甘氨酸。 29. A compound according to claim 27, wherein L1, L2 and L3 are each independently selected from: 6 :( sequence Gly) 3 Lys (Gly) 4; 7 :( sequence Gly) 3 Ser asparagine glycine (Gly) 2; 8 :( sequence Gly) 3 Cys (Gly) 4; and sequence 9: glycine, proline, asparagine glycine.
30.根据权利要求27的化合物,其中L1,L2或L3含有半胱氨酸残基。 30. The compound according to claim 27, wherein the L1, L2 or L3 comprises a cysteine ​​residue.
31.根据权利要求30的化合物的二聚体。 31. A dimer of the compound according to claim 30.
32.根据权利要求26的化合物,其中L1,L2或L3含有(CH2)n,其中n是1-20的数字。 32. A compound according to claim 26, wherein the L1, L2 or L3 comprises (the CH2) n, where n is a number from 1 to 20.
33.根据权利要求1-8中任一项的化合物,选自:序列22:Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA序列23:Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA-Fc序列24:IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA-Fc序列25:Fc-GG-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA序列26:Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA序列27:Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA,其为环状 33. A compound as claimed in any one of the claim 1 selected from: SEQ ID 22: Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA sequence 23: Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA-Fc sequence 24: IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA-Fc sequence 25: Fc-GG-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA sequence 26: Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA sequence 27: Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA, which is a cyclic 序列28:Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA,其为线性序列29:Fc-IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA序列30:Fc-IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA序列31:Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA序列32:Fc-IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA序列33:Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA|Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA序列34:Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA。 Sequence 28: Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA, which is a linear sequence of 29: Fc-IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA sequence 30: Fc-IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA sequence 31: Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA sequence 32: Fc-IEGPTLRQWLAARA -GGGNGSGG-IEGPTLRQWLAARA sequence 33: Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA | Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA sequence 34: Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA.
34.权利要求1-8中任一项的化合物在制备用于增加有此需要的患者的巨核细胞或血小板数量的药物中的用途。 Compound as claimed in any one of claims 34 for use in increasing the number of nucleated cells, or giant platelet drugs in need of a patient.
35.根据权利要求34的用途,其中化合物的剂量为1μg/kg-100mg/kg。 35. Use according to claim 34, wherein the dose of the compound of 1μg / kg-100mg / kg.
36.一种药用组合物,含有权利要求1-8中任一项的化合物和与其混合的可药用载体。 36. A pharmaceutical composition comprising a compound according to any one of 1-8 in admixture with a pharmaceutically acceptable carrier claims.
37.编码权利要求17化合物的多核苷酸。 17 37. A polynucleotide encoding a compound as claimed in claim.
38.编码权利要求22化合物的多核苷酸。 22 38. A polynucleotide encoding a compound as claimed in claim.
39.编码权利要求27化合物的多核苷酸。 27 39. A polynucleotide encoding a compound as claimed in claim.
40.编码权利要求31化合物的多核苷酸。 31 40. A polynucleotide encoding a compound as claimed in claim.
41.含有权利要求37-40中任一项的多核苷酸的载体。 41. The polynucleotide of claim comprising the vector of any one of 37-40.
42.含有权利要求41载体的宿主细胞。 42. A host cell containing the vector of claim 41.
43.生产权利要求17、22、27或31的化合物的方法,该方法包括在适宜的营养培养基中生长权利要求42的宿主细胞,并从所述细胞或营养培养基中分离所述化合物。 17,22,27 or 31 of a compound produced as claimed in claim 43, the method comprising in a suitable nutrient medium a host cell growth as claimed in claim 42, and isolating said compound from said cell or nutrient medium.
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